CN116904418A - yfdH gene mutant and application thereof in preparation of L-valine - Google Patents
yfdH gene mutant and application thereof in preparation of L-valine Download PDFInfo
- Publication number
- CN116904418A CN116904418A CN202311058879.0A CN202311058879A CN116904418A CN 116904418 A CN116904418 A CN 116904418A CN 202311058879 A CN202311058879 A CN 202311058879A CN 116904418 A CN116904418 A CN 116904418A
- Authority
- CN
- China
- Prior art keywords
- microorganism
- yfdh
- valine
- seq
- recombinant
- 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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 title claims abstract description 152
- 229960004295 valine Drugs 0.000 title claims abstract description 79
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 101150016378 yfdH gene Proteins 0.000 title abstract description 54
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 84
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 42
- 241000588724 Escherichia coli Species 0.000 claims abstract description 30
- 241000894006 Bacteria Species 0.000 claims abstract description 27
- 150000001413 amino acids Chemical group 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 claims abstract description 19
- 239000012620 biological material Substances 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims description 71
- 108020004414 DNA Proteins 0.000 claims description 66
- 102000053602 DNA Human genes 0.000 claims description 40
- 239000013598 vector Substances 0.000 claims description 37
- 230000014509 gene expression Effects 0.000 claims description 28
- 102000039446 nucleic acids Human genes 0.000 claims description 28
- 108020004707 nucleic acids Proteins 0.000 claims description 28
- 150000007523 nucleic acids Chemical class 0.000 claims description 28
- 210000004027 cell Anatomy 0.000 claims description 22
- 108091026890 Coding region Proteins 0.000 claims description 19
- 239000002773 nucleotide Substances 0.000 claims description 16
- 125000003729 nucleotide group Chemical group 0.000 claims description 16
- 238000010276 construction Methods 0.000 claims description 14
- 230000001105 regulatory effect Effects 0.000 claims description 12
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 8
- 239000004473 Threonine Substances 0.000 claims description 8
- 229960000310 isoleucine Drugs 0.000 claims description 8
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 8
- 235000013305 food Nutrition 0.000 claims description 6
- 239000002537 cosmetic Substances 0.000 claims description 4
- 125000000539 amino acid group Chemical group 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 102000037865 fusion proteins Human genes 0.000 claims description 2
- 108020001507 fusion proteins Proteins 0.000 claims description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims description 2
- 101100338056 Escherichia coli (strain K12) yfdH gene Proteins 0.000 abstract description 51
- 230000035772 mutation Effects 0.000 abstract description 33
- 238000009825 accumulation Methods 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 230000002018 overexpression Effects 0.000 abstract description 4
- 239000013612 plasmid Substances 0.000 description 44
- 239000012634 fragment Substances 0.000 description 35
- 235000018102 proteins Nutrition 0.000 description 28
- 238000000855 fermentation Methods 0.000 description 20
- 230000004151 fermentation Effects 0.000 description 20
- 238000012408 PCR amplification Methods 0.000 description 17
- 108091027544 Subgenomic mRNA Proteins 0.000 description 10
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000000137 annealing Methods 0.000 description 8
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 8
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 8
- 239000004474 valine Substances 0.000 description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 229960000268 spectinomycin Drugs 0.000 description 7
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 7
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000011426 transformation method Methods 0.000 description 5
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 4
- 108091033409 CRISPR Proteins 0.000 description 4
- 241000186226 Corynebacterium glutamicum Species 0.000 description 4
- 102000008300 Mutant Proteins Human genes 0.000 description 4
- 108010021466 Mutant Proteins Proteins 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 229940104302 cytosine Drugs 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229940113082 thymine Drugs 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 206010016256 fatigue Diseases 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000012257 pre-denaturation Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 238000010361 transduction Methods 0.000 description 3
- 230000026683 transduction Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 2
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000010354 CRISPR gene editing Methods 0.000 description 2
- 241000186216 Corynebacterium Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000319304 [Brevibacterium] flavum Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- -1 compound branched-chain amino acid Chemical class 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000006209 dephosphorylation reaction Methods 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 108010045262 enhanced cyan fluorescent protein Proteins 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 241000589159 Agrobacterium sp. Species 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 206010010075 Coma hepatic Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 241000572303 Corynebacterium pekinense Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 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
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-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
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-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
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 125000003580 L-valyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(C([H])([H])[H])(C([H])([H])[H])[H] 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 208000002847 Surgical Wound Diseases 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 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
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-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
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 150000001768 cations Chemical class 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
- 230000001684 chronic effect Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000013604 expression vector Substances 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
- 238000003209 gene knockout Methods 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
- 230000002394 glycogenic effect Effects 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 201000001059 hepatic coma Diseases 0.000 description 1
- 208000007386 hepatic encephalopathy Diseases 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 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 1
- 201000006370 kidney failure Diseases 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
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- JXOHGGNKMLTUBP-HSUXUTPPSA-N shikimic acid Chemical compound O[C@@H]1CC(C(O)=O)=C[C@@H](O)[C@H]1O JXOHGGNKMLTUBP-HSUXUTPPSA-N 0.000 description 1
- JXOHGGNKMLTUBP-JKUQZMGJSA-N shikimic acid Natural products O[C@@H]1CC(C(O)=O)=C[C@H](O)[C@@H]1O JXOHGGNKMLTUBP-JKUQZMGJSA-N 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/175—Amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Pharmacology & Pharmacy (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Birds (AREA)
- Nutrition Science (AREA)
- Animal Husbandry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Dermatology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses a yfdH gene mutant and application thereof in preparation of L-valine, and belongs to the technical field of biology. The technical problem to be solved is how to increase the yield of the L-valine of the escherichia coli. In order to solve the technical problem, the application provides a protein with an amino acid sequence shown as SEQ ID No.4, a coding gene thereof, related biological materials and application thereof in improving the yield of L-valine. The application constructs the over-expression mutant yfdH T743C Recombinant bacteria of the gene or the wild yfdH gene, and point mutation recombinant bacteria, and experimental results show that: overexpression of yfdH Gene or yfdH T743C The gene can obviously improve the yield of L-valine. Point mutation (T-C) of yfdH gene also contributes to the improvement of L-valine production. The genetic engineering strain constructed by the method can obviously promote accumulation of L-valine and improve the yield of L-valine.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a yfdH gene mutant and application thereof in preparation of L-valine.
Background
L-Valine (L-Valine) is a branched nonpolar alpha-amino acid containing five carbon atoms, is an essential amino acid and a glycogenic amino acid for mammals, and cannot be synthesized by humans and animals themselves. L-valine has effects of promoting protein synthesis, inhibiting protein decomposition, enhancing immunity, and correcting negative nitrogen balance caused by operation, wound, infection, etc. In addition, the L-valine also has the effects of resisting central fatigue, resisting peripheral fatigue, delaying exercise fatigue and accelerating the restoration of a body after exercise, so that the L-valine has wide application and commercial value in the industries of animal feed, cosmetics, foods and medicines. The compound branched-chain amino acid transfusion prepared from the L-valine has wide application in the treatment of blood brain barrier, hepatic coma, chronic liver cirrhosis and renal failure, the dietary treatment of congenital metabolic defect, the treatment of septicemia and postoperative diabetes patients, the treatment for accelerating the healing of surgical wounds and the nutritional support treatment of tumor patients. L-valine is mainly used as a food additive, a nutrient supplement liquid, a flavoring agent and the like in the food industry. The L-valine gel has positively charged terminal groups, is a novel low molecular weight gel, can be prepared to form hydrogel, and has been widely applied in the fields of biological medicine, tissue engineering, photochemistry, electrochemistry, food industry, cosmetics and the like.
At present, the production method of L-valine mainly comprises an extraction method, a chemical synthesis method and a fermentation method. The extraction method and the chemical synthesis method have the advantages of limited sources of raw materials, high production cost, low yield, serious pollution and difficult realization of industrial production. The microorganism direct fermentation method for producing L-valine has the advantages of wide raw material source, low cost, mild reaction condition, easy realization of large-scale production and the like, and is a very economical and efficient production method. The strain with high yield obtained in industrial fermentation is of great importance for the fermentation production of L-valine, and is a core of the whole L-valine fermentation industry and an important factor for determining the industrial value of fermentation products. Along with the continuous development of genetic engineering breeding technology, the production bacteria are modified from the molecular level, and the functions of related genes are researched and excavated, thus providing a wide prospect for the industrialized fermentation production of L-valine. Therefore, the breeding of high-yield and stable production strains promotes the accumulation of L-valine in microorganisms, and further improves the yield of L-valine, thereby being beneficial to promoting the industrialization process of L-valine.
Disclosure of Invention
The technical problem to be solved by the present application is how to increase the yield of L-valine in E.coli by genetic engineering of genes, and the technical problem to be solved is not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
To solve the technical problems, the application firstly provides protein which can be named YfdH I248T The protein may comprise any one of the following:
a1 Amino acid sequence comprising a protein as shown in SEQ ID No. 4;
a2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.4, has more than 90% of identity with the protein shown in A1) and has the same function;
a3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).
Labels described herein include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, the 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
The application also provides nucleic acid molecules, which may be named yfdH T743C The nucleic acid molecule may comprise any one of the following:
b1 Encoding the protein YfdH I248T Is a nucleic acid molecule of (a);
b2 The coding sequence is a DNA molecule shown as SEQ ID No. 3.
The DNA molecule shown in SEQ ID No.3 is the mutant gene yfdH of the application T743C 。
The DNA molecule shown in SEQ ID No.3 (yfdH T743C Gene) encodes a mutant protein YfdH shown in SEQ ID No.4 I248T 。
The yfdH T743C The 743 rd cytosine (C) in the nucleotide sequence (SEQ ID No. 3) of the gene is mutated from thymine (T), the protein YfdH I248T Threonine (T) at position 248 in the amino acid sequence (SEQ ID No. 4) is mutated from isoleucine (I).
The present application also provides a biomaterial that may include any one of the following:
c1 Containing said nucleic acid molecule yfdH T743C Is a gene expression cassette;
c2 Containing said nucleic acid molecule yfdH T743C Or a recombinant vector comprising the expression cassette of C1);
c3 Containing said nucleic acid molecule yfdH T743C Or a recombinant microorganism comprising the expression cassette of C1) or a recombinant microorganism comprising the recombinant vector of C2);
c4 Containing said nucleic acid molecule yfdH T743C Or a recombinant cell comprising the expression cassette of C1) or a recombinant cell comprising the recombinant vector of C2).
Further, C3) the recombinant microorganism may be any one of the following:
d1 A recombinant microorganism obtained by introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 into a microorganism of interest;
d2 A recombinant microorganism obtained by introducing a DNA molecule having a coding sequence represented by SEQ ID No.3 into a microorganism of interest;
d3 A recombinant microorganism obtained by mutating the nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest to C.
The application also provides the protein YfdH I248T Said nucleic acid molecule yfdH T743C Or any one of the following applications of the biomaterial:
e1 Use in regulating the production of microbial L-valine;
e2 Use in the preparation of L-valine;
e3 The application of the strain in constructing genetically engineered bacteria for producing L-valine.
The modulation described herein may be either an increase (up-regulation) or a decrease (down-regulation).
Further, the modulation of L-valine production by a microorganism as described herein can be an increase (up-regulation) or a decrease (down-regulation) in the accumulation of L-valine in the microorganism (i.e., promotion or inhibition of L-valine biosynthesis).
The application also provides an application of any one of F1) -F4) in regulating and controlling the yield of L-valine of microorganisms, preparing L-valine or constructing genetically engineered bacteria producing L-valine, wherein the F1) -F4) is as follows:
f1 Amino acid sequence comprising a protein as shown in SEQ ID No. 2;
f2 A nucleic acid molecule encoding the protein of F1);
f3 A DNA molecule with a coding sequence shown in SEQ ID No. 1;
f4 An expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell containing F2) said nucleic acid molecule or F3) said DNA molecule.
The DNA molecule shown in SEQ ID No.1 is also the yfdH gene of the present application.
The DNA molecule shown in SEQ ID No.1 (yfdH gene) encodes the protein YfdH shown in SEQ ID No. 2.
The present application also provides a method for increasing the yield of L-valine or producing L-valine of a microorganism of interest, which can comprise any one of the following:
g1 Increasing the nucleic acid molecule yfdH in a microorganism of interest T743C The expression amount or content of (a) to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g2 Increasing the expression level or the content of F2) the nucleic acid molecule or F3) the DNA molecule in the microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g3 A DNA molecule having a nucleotide sequence of SEQ ID No.1 in a microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest, the mutation may comprise: isoleucine at position 248 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 was mutated to threonine.
Further, the G1) may be achieved by any one of the following methods: (1) Increasing yfdH in the microorganism of interest T743C Copy number of Gene (SEQ ID No. 3) (e.g., yfdH to be single-or multiple-copy T743C Gene transfer into the microorganism of interest); (2) Enhancement of yfdH by expression control sequences on the basis of (1) T743C Expression of genes (e.g. modified yfdH T743C Expression control sequences of the genes).
Further, the G2) may be achieved by any one of the following methods: (1) Increasing the copy number of the yfdH gene (SEQ ID No. 1) in the microorganism of interest (e.g., introducing a single copy or multiple copies of the yfdH gene into the microorganism of interest); (2) The expression of the yfdH gene is enhanced by an expression control sequence (e.g., the expression control sequence of the yfdH gene is modified).
The expression control sequence may be a promoter, enhancer or silencer sequence.
Further, the mutation in G3) may be performed by a gene editing technique, a homologous recombination technique, or a site-directed mutagenesis, etc.
In the above method, the microorganism of interest may be Escherichia coli.
In the above method, the method may include any one of the following:
h1 Introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 and/or SEQ ID No.2 into the microorganism of interest;
h2 Introducing a DNA molecule comprising the coding sequence shown as SEQ ID No.3 and/or SEQ ID No.1 into the microorganism of interest;
h3 The nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest is mutated to C.
The application also provides the protein YfdH I248T Said nucleic acid molecule yfdH T743C Use of said biological material, a protein as described in F1) herein, a nucleic acid molecule as described in F2) herein, a DNA molecule as described in F3) herein, an expression cassette as described in F4) herein, a recombinant vector, a recombinant microorganism or a recombinant cell for the preparation of a food, a cosmetic, a pharmaceutical or a feed comprising L-valine.
The vectors described herein refer to vectors capable of carrying exogenous DNA or genes of interest into host cells for amplification and expression, and may be cloning vectors or expression vectors, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), or viral vectors.
The microorganism described herein may be a yeast, bacterium, algae or fungus. The bacteria may be derived from, but not limited to, escherichia sp, erwinia sp, agrobacterium sp, flavobacterium sp, alcaligenes sp, pseudomonas sp, bacillus sp, brevibacterium sp, corynebacterium sp, aerobacter sp, enterobacter sp, micrococcus sp, serratia sp, salmonella sp, streptomyces sp, provicum sp, and the like.
Further, the bacterium may be Escherichia coli (Escherichia coli), corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium lactofermentum (Brevibacterium lactofermentum), brevibacterium flavum (Brevibacterium flavum), or Corynebacterium beijing (Corynebacterium pekinense).
The cells described herein may be plant cells or animal cells. The cell may be any biological cell that can synthesize the amino acid of interest.
The recombinant vector described herein may be recombinant vector pSTV28-yfdH and/or recombinant vector pSTV28-yfdH T743C 。
The recombinant vector pSTV28-yfdH contains the DNA molecule shown in SEQ ID No.6 (i.e., the yfdH gene and regulatory element fragment). The DNA molecule shown in SEQ ID No.6 comprises a promoter P J23105 RBS sequence of pTrc99a plasmid, wild-type yfdH gene, terminator Trc. Promoter P J23105 And RBS sequences were designed in primer P1 (SEQ ID No. 8) and primer P6 (SEQ ID No. 13), and terminator Trc was designed in primer P4 (SEQ ID No. 11) and primer P5 (SEQ ID No. 12).
The recombinant vector pSTV28-yfdH T743C Contains the DNA molecule shown in SEQ ID No.7 (i.e., yfdH T743C Gene and regulatory element fragments). The DNA molecule shown in SEQ ID No.7 comprises a promoter P J23105 RBS sequence, yfdH of pTrc99a plasmid T743C Gene, terminator Ttrc. Promoter P J23105 And RBS sequences were designed in primer P1 (SEQ ID No. 8) and primer P6 (SEQ ID No. 13), and terminator Trc was designed in primer P4 (SEQ ID No. 11) and primer P5 (SEQ ID No. 12).
The recombinant microorganism described herein may be VHY (overexpressing the yfdH gene), VHY (overexpressing yfdH) T743C Gene), VHY (yfdH) T743C Point mutation), MG1655-02 (overexpressing the yfdH gene), MG1655-03 (overexpressing the yfdH gene) T743C Gene) and/or MG1655-04 (yfdH) T743C Point mutations).
The recombinant microorganisms VHY and VHY are the recombinant vectors pSTV28-yfdH and pSTV28-yfdH T743C Recombinant microorganisms obtained by introducing E.coli VHY.
The recombinant microorganisms MG1655-02 and MG1655-03 were prepared by subjecting the recombinant vectors pSTV28-yfdH and pSTV28-yfdH T743C Recombinant microorganisms obtained by introducing E.coli MG1655, respectively.
The recombinant microorganism VHY is obtained by mutating the nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the escherichia coli VHY into C.
The recombinant microorganism MG1655-04 is obtained by mutating a nucleotide T at 743 of a DNA molecule shown in SEQ ID No.1 in a genome of Escherichia coli MG1655 into C.
The introduction may be a transformation of a vector carrying the DNA molecule of the present application into a host bacterium by any known transformation method such as a chemical transformation method (e.g., ca2+ -induced transformation method, polyethylene glycol-mediated transformation method or metal cation-mediated transformation method) or electroporation transformation method; the DNA molecules of the application may also be transduced into host bacteria by phage transduction. The DNA molecules to be introduced may be either single or multiple copies. The introduction may be by integrating the exogenous gene into the host chromosome or by extrachromosomal expression from a plasmid.
The method for producing L-valine described herein may be a fermentation method for producing L-valine, and specifically may be a fermentation method for producing L-valine by using the recombinant microorganisms VHY, VHY, VHY24, MG1655-02, MG1655-03 and/or MG 1655-04.
The microorganism of interest described herein may be E.coli (Escherichia coli), and specifically E.coli VHY or E.coli MG1655.
Using the yfdH gene or a variant thereof (e.g., yfdH T743C Genes) can also be used to produce a variety of products including, but not limited to, lysine, glutamic acid, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha ketoglutaric acid, citric acid, ornithine and/or citrulline.
The application constructs the over-expression yfdH by taking the escherichia coli MG1655 and the escherichia coli VHY as starting bacteria T743C Recombinant strain of gene (SEQ ID No. 3) or yfdH gene (SEQ ID No. 1), and yfdH gene against Escherichia coli MG1655 and Escherichia coli VHY20The point mutation is carried out, recombinant bacteria with the point mutation are constructed, and fermentation experiments are carried out on the constructed engineering strain, and the result shows that: overexpression of the yfdH gene or yfdH in either the L-valine-producing strain (e.g., VHY) or the wild-type E.coli (e.g., MG 1655) T743C The genes all contribute to the synthesis of L-valine and over-express yfdH T743C The gene can remarkably improve the yield of L-valine. Furthermore, in either L-valine-producing strain or wild-type E.coli, the yfdH gene was subjected to a point mutation, and after the T at position 743 was mutated to C (the mutant protein YfdH was obtained accordingly) I248T Mutation of isoleucine to threonine at position 248 of the amino acid sequence) contributes to a significant increase in L-valine production. Meanwhile, the expression of the deletion yfdH gene is unfavorable for the synthesis of L-valine. Since the sugar supplement amounts are the same, an increase in yield means an increase in conversion. The genetic engineering strain constructed by the application can obviously promote accumulation of L-valine and improve the yield of L-valine. The application cultivates high-yield and high-quality strains which accord with industrial production, and is beneficial to promoting the industrial production process of L-valine.
Drawings
FIG. 1 shows the composition of L-valine fermentation medium in example 7.
FIG. 2 shows the results of fermentation experiments for L-valine in example 7.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The pSTV28 plasmid in the following examples is a Bao Bio Inc., cat: 3331.
coli MG1655 in the following examples was derived from beijing na-invasive alliance biotechnology institute (BNCC), strain number: BNCC363342.
The following examples are described in E.coli VHY: wang Jiachu, wu Faqing, wu Heyun, et al. Engineering strains for the production of valine by oxygen-limited fermentation and optimization of the fermentation process [ J ]. Food and fermentation industry, 2023,49 (1): 8.
The pREDCas9 plasmid and pGRB vector in the examples below are products of the addgene company.
Example 1 construction of a coding region fragment of yfdH Gene and wild-type yfdH Gene comprising Point mutations
According to NCBI published Escherichia coli (Escherichia coli) MG1655 genome sequence, two pairs of primers for amplifying the coding region of the yfdH gene were designed and synthesized, and a point mutation was introduced into the yfdH gene coding region (SEQ ID No. 1) of Escherichia coli MG1655 (it was confirmed by sequencing that the wild-type yfdH gene remained on the chromosome of the strain) in an allelic substitution manner, the point mutation being a mutation of thymine (T) at position 743 in the nucleotide sequence (SEQ ID No. 1) of the yfdH gene into cytosine (C), to give a DNA molecule shown in SEQ ID No.3 (mutated yfdH gene, which is named yfdH) T743C )。
Wherein the DNA molecule shown in SEQ ID No.1 encodes a protein having the amino acid sequence of SEQ ID No.2 (said protein is named protein YfdH).
The DNA molecule shown in SEQ ID No.3 encodes a mutein having the amino acid sequence of SEQ ID No.4 (said mutein is named YfdH I248T ). The mutant protein YfdH I248T Threonine (T) at position 248 in the amino acid sequence (SEQ ID No. 4) is mutated from isoleucine (I).
The mutant fragment was constructed by PCR, and the primer design was as follows (synthesized by Jin Wei, suzhou) with the bold base as the mutation position:
P1:5'-TAGGTACTATGCTAGCAGGAAACAGACCATGAAGATATCTCTTGTAGTTCC-3' (underlined nucleotide sequence contains the gene regulatory element sequence) (SEQ ID No. 8),
P2:
P3:
P4:5'-CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTT GTCATTTTTTGACTCTCTTGATG-3' (underlined nucleotide sequence contains the gene regulatory element sequence) (SEQ ID No. 11).
The construction method comprises the following steps: using Escherichia coli MG1655 as a template, and performing PCR amplification with primers P1 and P2, P3 and P4, respectively, to obtain two DNA fragments (yfdH) each having a coding region of the yfdH gene having a mutation base and a length of 784bp and 230bp, respectively T743C -Up and yfdH T743C Down). The Escherichia coli MG1655 is used as a template, and primers P1 and P4 are used for PCR amplification to obtain a DNA fragment with the length of 988bp and containing the coding region of the yfdH gene.
The PCR amplification system is as follows: 5 XPrimeSTAR Buffer (Mg) 2+ Plus) 10. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, primer (10 pM) 2. Mu.L each, template<200 ng), primeSTAR HS DNA Polymerase (2.5U/. Mu.L) 0.5. Mu.L, sterile water was added to a total volume of 50. Mu.L.
The PCR amplification reaction procedure was: pre-denaturation at 98℃for 10sec, (denaturation at 95℃for 5min; annealing at 55℃for 30s; extension at 72℃for 60sec;30 cycles), over-extension at 72℃for 10min.
Two DNA fragments (yfdH T743C -Up and yfdH T743C Down) is separated and purified by agarose gel electrophoresis, and then subjected to yfdH T743C -Up and yfdH T743C Overlapping PCR amplification is carried out by using the primer P1 and the primer P4 by taking Down as a template to obtain the primer containing yfdH with the length of 988bp T743C DNA fragments of the coding region of the gene.
Example 2 construction of overexpressed yfdH Gene or yfdH T743C Recombinant vector of gene
PCR was used to construct pSTV28 plasmid linearized vector fragment, and the primer design was as follows (synthesized by Jin Wei, suzhou):
P5:5'-AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA TGTGAGCGAGGAAGCGGAATA-3' (underlined nucleotide sequence comprising the sequence of the gene regulatory element) (SEQ ID No. 12),
P6:5'-GCTAGCATAGTACCTAGGACTGAGCTAGCCGTAAAGAAGATGCCAGGAAGATACTTAACA-3' (underlined nucleotide sequence contains the gene regulatory element sequence) (SEQ ID No. 13).
The construction method comprises the following steps: the pSTV28 plasmid (containing chloramphenicol resistance marker) is used as a template, and primers P5 and P6 are used for PCR amplification to obtain a pSTV28 plasmid linearization vector fragment with the length of 3027bp, and the pSTV28 plasmid linearization vector fragment is separated and purified by agarose gel electrophoresis for later use. The nucleotide sequence of the pSTV28 plasmid linearized vector fragment is shown in SEQ ID No. 5.
The DNA fragment (988 bp, without point mutation) containing the coding region of the yfdH gene constructed in example 1 and the gene containing yfdH were subjected to T743C The DNA fragment (988 bp, containing point mutation) of the gene coding region is respectively connected with the pSTV28 plasmid linearization vector fragment to obtain recombinant vectors, which are respectively named as: pSTV28-yfdH and pSTV28-yfdH T743C 。
Positive recombinant vector pSTV28-yfdH T743C Sequencing and identification of the recombinant vector pSTV28-yfdH containing the correct Point mutation (T-C) by the company Jin Weizhi, suzhou T743C And (5) storing for standby. Sequencing results showed that thymine (T) at position 743 of the coding region of yfdH gene was mutated to cytosine (C), ultimately resulting in mutation of isoleucine (I) at position 248 of the encoded protein to threonine (T).
The recombinant vector pSTV28-yfdH contains the DNA molecule shown in SEQ ID No.6 (i.e., the yfdH gene and regulatory element fragment). The DNA molecule shown in SEQ ID No.6 comprises a promoter P J23105 RBS sequence of pTrc99a plasmid, wild-type yfdH gene, terminator Trc. Promoter P J23105 And RBS sequences are designed in primer P1 and primer P6, and terminator Trc is designed in primer P4 and primer P5.
Recombinant vector pSTV28-yfdH T743C Contains the DNA molecule shown in SEQ ID No.7 (i.e., yfdH T743C Gene and regulatory element fragments). The DNA molecule shown in SEQ ID No.7 comprises a promoter P J23105 RBS sequence, yfdH of pTrc99a plasmid T743C Gene, terminator Ttrc.Promoter P J23105 And RBS sequences are designed in primer P1 and primer P6, and terminator Trc is designed in primer P4 and primer P5.
EXAMPLE 3 construction of plasmids containing pSTV28, pSTV28-yfdH T743C Is an engineered strain of (2)
The construction method comprises the following steps: plasmids pSTV28 and the plasmids of example 2 (pSTV 28-yfdH, pSTV28-yfdH T743C ) After electric transduction into E.coli VHY, the single colony generated by culture is identified by primer P7 (5'-CAGGGATTGGCTGAGACGAA-3', SEQ ID No. 14) and primer P8 (5'-TGTTAAGTATCTTCCTGGCATCTTC-3', SEQ ID No. 15), positive strains can amplify 1075bp, 1996bp and 1996bp size bands respectively. The positive strains were named VHY, VHY, VHY23, respectively. Recombinant VHY contained plasmid pSTV28; recombinant VHY contained plasmid pSTV28-yfdH; recombinant VHY contains plasmid pSTV28-yfdH T743C 。
EXAMPLE 4 construction of the plasmid containing pSTV28, pSTV28-yfdH in wild type E.coli MG1655 T743C Is an engineered strain of (2)
The construction method comprises the following steps: plasmids pSTV28 and the plasmids of example 2 (pSTV 28-yfdH, pSTV28-yfdH T743C ) After electric transduction into the wild type MG1655 of the escherichia coli respectively, the single colonies generated by culture are respectively identified by a primer P7 (SEQ ID No. 14) and a primer P8 (SEQ ID No. 15), and positive strains can respectively amplify 1075bp, 1996bp and 1996bp size bands. Positive strains were designated MG1655-01, MG1655-02, MG1655-03, respectively. Recombinant MG1655-01 contains plasmid pSTV28; recombinant MG1655-02 contained plasmid pSTV28-yfdH; recombinant MG1655-03 containing plasmid pSTV28-yfdH T743C 。
Example 5, wild-type E.coli MG1655 and valine-producing bacterium VHY yfdH T743C Construction of Point mutations
According to the genomic sequence of Escherichia coli MG1655 published by NCBI, thymine (T) at 743bp in the yfdH gene coding region of MG1655 and L-valine producing bacterium VHY is mutated into cytosine (C) by CRISPR/Cas9 gene editing technique,thus, the mutant yfdH was studied more intensively T473C Influence of the Gene on the amount of L-valine synthesized in wild-type E.coli and L-valine-producing bacterium VHY.
1. Construction of sgRNA plasmids
According to NCBI published Escherichia coli (Escherichia coli) MG1655 genome sequence, using CRISPRRGEN Tools (http:// www.rgenome.net/cas-designer /) to design the sgRNA target sequence, after selecting the appropriate sgRNA target sequence, linearization pGRB cloning vector terminal sequences were added at the 5 'and 3' extreme ends of the target sequence to form the complete sgRNA plasmid by recombination.
The amplification of the DNA fragment containing the sgRNA target sequence is carried out without a template and only by carrying out the PCR annealing process, and the system and the procedure are as follows. PCR reaction system: 10. Mu.L of sgRNA-F and 10. Mu.L of sgRNA-R; PCR reaction procedure: denaturation at 95℃for 5min and annealing at 50℃for 1min. After the completion of annealing, the target fragment (DNA fragment containing the sgRNA target sequence) was recovered using a DNA purification kit, the DNA concentration was determined, and the concentration was diluted to 100 ng/. Mu.L.
The pGRB plasmid was digested with SpeI to give a linearized pGRB plasmid, which was then dephosphorylated to prevent the pGRB plasmid from self-ligating. And (3) enzyme cutting system: 10 XBuffer 5. Mu.L, speI 2.5. Mu.L, pGRB plasmid DNA 3000-5000ng, ddH 2 O to 50. Mu.L. Enzyme digestion at 37 ℃ for 3 hours, agarose gel electrophoresis gel cutting recovery, dephosphorylation reaction and dephosphorylation system: 10 XBuffer 5. Mu.L, linearized pGRB plasmid DNA 1000-2000ng, CIAP 2.5. Mu.L, ddH 2 O to 50. Mu.L. After 1h treatment at 37℃the linearized pGRB plasmid was recovered using a DNA purification kit.
The DNA fragment containing the sgRNA target sequence was subjected to homologous recombination ligation with the linearized pGRB plasmid using the Gibson Assemblem kit (New England). Recombination system: NEB assembly enzyme 2.5. Mu.L, linearized pGRB plasmid 2. Mu.L, DNA fragment containing sgRNA target sequence 0.5. Mu.L. After 30min of assembly at 50 ℃, DH5 alpha competent cells are transformed from the product, plasmids are extracted, and sequencing primers sgRNA-PF/sgRNA-PR are used for sequencing and identification. The constructed sgRNA plasmid was designated pGRB-sgRNA-yfdH.
The primers used in this experiment were designed as follows (synthesized by Shanghai Invitrogen) with underlined bases as pGRB cloning vector homology arm sequence and lowercase bases as sgRNA target sequence:
sgRNA-F:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTactttggaatttagcacttgGTT TTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3'(SEQ ID No.16)，
sgRNA-R:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACcaagtgctaaattccaaagtACT AGTATTATACCTAGGACTGAGCTAGCTGTCA-3'(SEQ ID No.17)，
sgRNA-PF：5'-GTCTCATGAGCGGATACATATTTG-3'(SEQ ID No.18)，
sgRNA-PR：5'-ATGAGAAAGCGCCACGCT-3'(SEQ ID No.19)。
2. DNA fragment Up-yfdH of yfdH gene point mutation T743C Acquisition of Down
Two pairs of primers for amplifying the upstream and downstream homology arm sequences are designed and synthesized according to the genome sequence of Escherichia coli MG1655 published by NCBI, and T743C point mutation is introduced into yfdH genes of Escherichia coli MG1655 and valine producer VHY respectively in a CRISPR/Cas9 gene editing mode.
The primers were designed as follows (synthesized by the company epivitrogen, shanghai) with the bolded bases as mutation positions:
P9:5'-CCTGTCTTCAATGAAGAAGAAG-3'(SEQ ID No.20)，
P10:5'-AATATGATAGTATCTAAAGT CATCCACGCCCCATAAATAA-3'(SEQ ID No.21)，
P11:5'-TTATTTATGGGGCGTGGATGACTTTAGATACTATCATATT-3'(SEQ ID No.22)，
P12:5'-ACATTGACAGTGTTGATGAAG-3'(SEQ ID No.23)。
the genome DNA of MG1655 or valine producing bacterium VHY is used as a template, and primers P9/P10, P11/P12 and KAPA HiFi HotStart are used for PCR amplification, so that an upper homology arm fragment 740bp and a lower homology arm fragment 620bp are obtained. After the PCR reaction is finished, agarose gel electrophoresis recovery is respectively carried out by adopting a column type DNA gel recovery kit. The recovered DNA is used as a template, and primers P9 and P12 are used for overlap PCR amplification to obtain a DNA fragment Up-yfdH with yfdH gene point mutation T743C -Down 1320bp。
PCR amplification and overlap PCR amplificationThe system comprises: 5 XHiFi with Mg 2+ Buffer 10. Mu.L, dNTP mix (10 mM) 1.5. Mu.L, primers (10 pM) 1.6. Mu.L, KAPA HiFi HotStart (1U/. Mu.L) 0.5. Mu.L, and ddH were supplemented 2 O to a total volume of 50. Mu.L.
PCR amplification and overlap PCR amplification procedure: pre-denaturation at 95℃for 5min, (denaturation at 98℃for 20s; annealing at 56℃for 15s; extension at 72℃for 150s;30 cycles), over-extension at 72℃for 5min.
3. Preparation and transformation of competent cells
pREDCas9 plasmid (containing spectinomycin resistance gene) is respectively transformed into MG1655 and valine producer VHY competent cells, the cells are spread on a 2-YT agar plate containing spectinomycin (100 MG/L) for culture at 32 ℃, single colony of the spectinomycin (100 MG/L) is selected and PCR identification is carried out by taking pRedCas9-PF/pRedCas9-PR as a primer and r Taq, so that 943bp MG1655-Cas9 and VHY-Cas 9 transformants containing pREDCas9 plasmid are obtained.
Preparation of MG1655-Cas9, VHY20-Cas9 competent cells when the thallus grows to OD 600 =0.1 IPTG was added at a final concentration of 0.1mM to induce λ -Red mediated homologous recombination. When OD is 600 When=0.6, cells were collected to prepare competent cells, and pGRB-sgRNA-yfdH plasmid and Up-yfdH plasmid were transformed, respectively T743C Down fragment, spread onto 2-YT agar plates containing spectinomycin (100 mg/L) and ampicillin (100 mg/L) and incubated at 32 ℃. And (3) carrying out PCR identification on single colonies generated by culture through a primer P13/P14 by using rTaq, carrying out PCR amplification to obtain a fragment with 660bp, sequencing, and comparing sequencing results to obtain positive bacteria with T743C point mutation.
Positive transformants were inoculated into 2-YT medium containing spectinomycin (100 mg/L) and arabinose at a final concentration of 0.2% to eliminate the plasmid pGRB-sgRNA-yfdH, colonies that grew on spectinomycin (100 mg/L) but did not grow on ampicillin (100 mg/L) were selected, these colonies were transferred to 2-YT medium for 42℃cultivation to eliminate pREDCas9 plasmid, colonies that did not grow on spectinomycin (100 mg/L) but grew on nonreactive 2-YT were selected, identified again by primer P13/P14 using rTaq PCR, PCR amplified to contain a 660bp fragment, and sequenced to produce a positive transformant with a T743C point mutation.
Gene editing of MG1655 was performed to obtain a mutant yfdH containing the point mutation T473C Recombinant strain (MG 1655/yfdH) T473C ) Designated MG1655-04, was obtained by gene editing of valine-producing bacterium VHY, and was found to contain the point mutation yfdH T473C Recombinant bacterium (VHY/yfdH) T473C ) Named VHY.
Recombinant MG1655-04 and VHY24 genome each containing point mutated yfdH T743C And (3) a gene.
Primers were designed as follows (synthesized by the company epivitrogen, shanghai):
P13:5'-AAATGGCAAGCAGGTGCTG-3'(SEQ ID No.24)，
P14:5'-CTTCAATTAATGGCTTAATG-3'(SEQ ID No.25)，
pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3'(SEQ ID No.26)，
pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3'(SEQ ID No.27)。
PCR amplification system: 2 XPromix Taq 12.5. Mu.L, 1. Mu.L each of primer (10 pM), and ddH was added 2 The total volume of O was 25. Mu.L.
PCR amplification procedure: pre-denaturing for 5min at 94℃and denaturing for 30s at 94 ℃; annealing at 56 ℃ for 30s; extending at 72 ℃ for 90s (30 cycles), and overextensing at 72 ℃ for 10min.
EXAMPLE 6 construction of genetically engineered bacterium MG1655. Delta. YfdH and VHY. Delta. YfdH
The genome of E.coli MG1655 or VHY was used as a template, and an upstream homology arm primer (UP-yfdH-S, UP-yfdH-A) and a downstream homology arm primer (DN-yfdH-S, DN-yfdH-A) were designed based on the sequence upstream and downstream of the coding region of the yfdH gene. PCR amplification was performed with primers UP-yfdH-S/UP-yfdH-A and DN-yfdH-S/DN-yfdH-A and KAPA HiFi HotStart to obtain an upper homology arm fragment 461bp and a lower homology arm fragment 898bp. After the PCR reaction is finished, agarose gel electrophoresis recovery is respectively carried out by adopting a column type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR amplification with primers UP-yfdH-S and DN-yfdH-A to obtain a DNA fragment of UP-delta yfdH-Down 1359bp from which the yfdH gene was knocked out.
PCR amplification and overlap PCR amplification system: 5 XHiFi with Mg 2+ Buffer 10. Mu.L, dNTP mix (10 mM) 1.5. Mu.L, primers (10 pM) 1.6. Mu.L, KAPA HiFi HotStart (1U/. Mu.L) 0.5. Mu.L eachL, supplement ddH 2 O to a total volume of 50. Mu.L.
PCR amplification and overlap PCR amplification procedure: pre-denaturation at 95℃for 5min, (denaturation at 98℃for 20s; annealing at 56℃for 15s; extension at 72℃for 150s;30 cycles), over-extension at 72℃for 5min.
Construction of pGRB-yfdH: the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-yfdH-S and gRNA-yfdH-A.
Preparation of competent cells of MG1655 or valine-producing bacterium VHY, construction of engineering strains MG1655 having reduced expression strength of yfdH Gene. Delta. YfdH and VHY: Δyfdh. When the PCR is verified, the length of the PCR amplified fragment of the positive bacterium is 1359bp, and the length of the PCR amplified fragment of the original bacterium is 1842bp. The resulting MG1655 was designated as MG1655-05; the resulting VHY20: Δyfdh is designated VHY25.MG1655-05 is a strain in which the yfdH gene is deleted in the genome of MG 1655; VHY 25A strain deleted of yfdH gene in the genome of VHY.
The primer sequences are shown below:
primer UP-yfdH-S:5'-TTTGTGTGTATAAGTTTTGTC-3' (SEQ ID No. 28),
primer UP-yfdH-A:5'-ATGATACTTTTATTGCTTTATTTCGCATCCCTAAAGACAATG-3' (SEQ ID No. 29),
primer DN-yfdH-S:5'-CATTGTCTTTAGGGATGCGAAATAAAGCAATAAAAGTATCAT-3' (SEQ ID No. 30),
primer DN-yfdH-A:5'-AAAAGAGTGCATTCGAAAACG-3' (SEQ ID No. 31),
primer gRNA-yfdH-S:5'-TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTACTTTGGAATTTAGCACTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG-3' (SEQ ID No. 32),
primer gRNA-yfdH-A:5'-CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCAAGTGCTAAATTCCAAAGTACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (SEQ ID No. 33).
Example 7L-valine fermentation experiment
Strains VHY (empty vector control), VHY (overexpressing yfdH gene), VHY23 (overexpressing yfdH) constructed in the above examples were used T743C Gene), VHY (yfdH) T743C Point mutation), VHY (deletion of yfdH Gene), MG1655-01 (empty vector control)) MG1655-02 (overexpressing the yfdH gene), MG1655-03 (overexpressing the yfdH gene) T743C Gene), MG1655-04 (yfdH) T743C Point mutations), MG1655-05 (deletion of the yfdH gene) and control strains VHY, MG1655 were subjected to shake flask fermentation test in a shaking incubator model ZQZY-CS8T (available from Shanghai know Chu instruments Co., ltd.). The fermentation test was performed at 37℃and 80rpm using the medium shown in FIG. 1, and each strain was repeated three times, and the results are shown in FIG. 2. Wherein the seed solution is cultured for 12 hours by using LB culture medium, and the fermentation inoculation amount is 10 percent (27 mL fermentation liquid and 3mL seed liquid).
As shown in FIG. 2, the yields of the over-expressed and point mutant genetically engineered bacteria (VHY, VHY, VHY24, MG1655-02, MG1655-03 and MG 1655-04) constructed by the application are obviously improved, and the yields of recombinant bacteria (VHY and MG 1655-05) after gene knockout are reduced, wherein compared with the yield of VHY of a control group, the yield of VHY is 16.23g/L, and the yield is obviously improved by 19.96%. At the same time, the yield (13.53 g/L) of strain VHY (empty vector control) was not significantly different from that of the blank VHY (13.24 g/L).
As can be seen from the above fermentation results, the yfdH gene or yfdH was overexpressed in either the L-valine-producing strain (e.g., VHY) or the wild-type E.coli (e.g., MG 1655) T743C The genes all contribute to the synthesis of L-valine and over-express yfdH T743C The gene can remarkably improve the yield of L-valine. Furthermore, in either L-valine-producing strain or wild-type E.coli, the yfdH gene was subjected to a point mutation, and after the T at position 743 was mutated to C (the mutant protein YfdH was obtained accordingly) I248T Mutation of isoleucine to threonine at position 248 of the amino acid sequence) contributes to a significant increase in L-valine production. Meanwhile, the expression of the deletion yfdH gene is unfavorable for the synthesis of L-valine. Since the sugar supplement amounts are the same, an increase in yield means an increase in conversion.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. A protein, characterized in that the protein comprises any one of the following:
a1 Amino acid sequence comprising a protein as shown in SEQ ID No. 4;
a2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.4, has more than 90% of identity with the protein shown in A1) and has the same function;
a3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).
2. A nucleic acid molecule, characterized in that the nucleic acid molecule comprises any one of the following:
b1 A nucleic acid molecule encoding the protein of claim 1;
b2 The coding sequence is a DNA molecule shown as SEQ ID No. 3.
3. A biomaterial, characterized in that the biomaterial comprises any one of the following:
c1 An expression cassette comprising the nucleic acid molecule of claim 2;
c2 A recombinant vector comprising the nucleic acid molecule of claim 2, or a recombinant vector comprising the expression cassette of C1);
c3 A recombinant microorganism comprising the nucleic acid molecule of claim 2, or a recombinant microorganism comprising the expression cassette of C1), or a recombinant microorganism comprising the recombinant vector of C2);
c4 A recombinant cell comprising the nucleic acid molecule of claim 2, or a recombinant cell comprising the expression cassette of C1), or a recombinant cell comprising the recombinant vector of C2).
4. A biomaterial according to claim 3 wherein C3) the recombinant microorganism is any one of the following:
d1 A recombinant microorganism obtained by introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 into a microorganism of interest;
d2 A recombinant microorganism obtained by introducing a DNA molecule having a coding sequence represented by SEQ ID No.3 into a microorganism of interest;
d3 A recombinant microorganism obtained by mutating the nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest to C.
5. Use of the protein of claim 1, the nucleic acid molecule of claim 2 or any of the following of the biological material of claim 3 or 4:
e1 Use in regulating the production of microbial L-valine;
e2 Use in the preparation of L-valine;
e3 The application of the strain in constructing genetically engineered bacteria for producing L-valine.
Use of any one of F1) -F4) in regulating the yield of L-valine in a microorganism, in the preparation of L-valine or in the construction of a genetically engineered bacterium producing L-valine, wherein the F1) -F4) is:
f1 Amino acid sequence comprising a protein as shown in SEQ ID No. 2;
f2 A nucleic acid molecule encoding the protein of F1);
f3 A DNA molecule with a coding sequence shown in SEQ ID No. 1;
f4 An expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell containing F2) said nucleic acid molecule or F3) said DNA molecule.
7. A method for increasing the production of L-valine or producing L-valine of a microorganism of interest, comprising any one of the following:
g1 Increasing the expression level or the content of the nucleic acid molecule according to claim 2 in a microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g2 Increasing the expression level or the content of the nucleic acid molecule F2) or the DNA molecule F3) in the microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest;
g3 A DNA molecule having a nucleotide sequence of SEQ ID No.1 in a microorganism of interest to obtain a microorganism having a higher L-valine yield than the microorganism of interest, comprising: isoleucine at position 248 of the amino acid sequence encoded by the DNA molecule shown in SEQ ID No.1 was mutated to threonine.
8. The method of claim 7, wherein the microorganism of interest is escherichia coli.
9. The method according to claim 7 or 8, characterized in that the method comprises any one of the following:
h1 Introducing a gene encoding a protein having an amino acid sequence represented by SEQ ID No.4 and/or SEQ ID No.2 into the microorganism of interest;
h2 Introducing a DNA molecule comprising the coding sequence shown as SEQ ID No.3 and/or SEQ ID No.1 into the microorganism of interest;
h3 The nucleotide T at 743 of the DNA molecule shown in SEQ ID No.1 in the genome of the microorganism of interest is mutated to C.
10. Use of the protein of claim 1, the nucleic acid molecule of claim 2, the biological material of claim 3 or 4, the protein of claim 6, the nucleic acid molecule, the DNA molecule, the expression cassette, the recombinant vector, the recombinant microorganism or the recombinant cell for the preparation of a food, a cosmetic, a pharmaceutical or a feed comprising L-valine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311058879.0A CN116904418A (en) | 2023-08-22 | 2023-08-22 | yfdH gene mutant and application thereof in preparation of L-valine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311058879.0A CN116904418A (en) | 2023-08-22 | 2023-08-22 | yfdH gene mutant and application thereof in preparation of L-valine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116904418A true CN116904418A (en) | 2023-10-20 |
Family
ID=88363119
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311058879.0A Pending CN116904418A (en) | 2023-08-22 | 2023-08-22 | yfdH gene mutant and application thereof in preparation of L-valine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116904418A (en) |
-
2023
- 2023-08-22 CN CN202311058879.0A patent/CN116904418A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113667682B (en) | YH66-RS11190 gene mutant and application thereof in preparation of L-valine | |
| WO2023231547A1 (en) | Ncgl2747 gene mutant and use thereof in preparation of l-lysine | |
| WO2022143763A1 (en) | Strain having enhanced l-glutamic acid productivity, construction method therefor and application thereof | |
| JP2024505626A (en) | L-glutamic acid producing recombinant strain with modified gene BBD29_04920, its construction method and application | |
| CN114181288B (en) | Process for producing L-valine, gene used therefor and protein encoded by the gene | |
| CN114317583B (en) | Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule used in method | |
| WO2008088149A1 (en) | Corynebacterium glutamicum variety producing l-arginine and method for fabricating the same | |
| CN114540399B (en) | Method for preparing L-valine, and gene mutant and biological material used by same | |
| CN114349831B (en) | aspA gene mutant, recombinant bacterium and method for preparing L-valine | |
| CN110846333A (en) | Recombinant strain modified by deoB gene and construction method and application thereof | |
| CN113563436B (en) | Engineering bacterium obtained by modification of YH66-RS03880 gene and application thereof in valine preparation | |
| CN116904418A (en) | yfdH gene mutant and application thereof in preparation of L-valine | |
| CN115820706A (en) | Galactose-1-uridine phosphate acyltransferase mutant and application thereof in preparation of L-lysine | |
| CN110804617B (en) | KdtA gene modified recombinant strain and construction method and application thereof | |
| CN116463304B (en) | Threonine dehydrogenase gene mutant and application thereof | |
| CN114277069B (en) | Method for preparing L-valine and biological material used by same | |
| CN112522175A (en) | Recombinant strain for producing L-glutamic acid by modifying gene BBD 29-09525 as well as construction method and application thereof | |
| CN114292315B (en) | Ppc mutant and application thereof in preparation of L-valine | |
| CN114410615B (en) | YH66_00525 protein and application of encoding gene thereof in regulating and controlling bacterial arginine yield | |
| CN114539367B (en) | CEY17_RS11900 gene mutant and application thereof in preparation of L-valine | |
| CN114540262B (en) | Method for constructing recombinant microorganism producing L-valine and nucleic acid molecule and biological material used in same | |
| CN116536237B (en) | Modified escherichia coli and application thereof in fermentation production of L-valine | |
| CN114907459B (en) | Engineering bacterium for high-yield arginine and construction method and application thereof | |
| CN114315998B (en) | CEY17_RS00300 gene mutant and application thereof in preparation of L-valine | |
| CN116555150B (en) | Recombinant Escherichia coli for fermentative production of L-valine |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20240315 Address after: 750100 Yang Yang Industrial Park, Yongning County, the Ningxia Hui Autonomous Region, Yinchuan Applicant after: NINGXIA EPPEN BIOTECH Co.,Ltd. Country or region after: China Address before: No.9, 13th Street, economic and Technological Development Zone, Binhai New Area, Tianjin Applicant before: TIANJIN University OF SCIENCE AND TECHNOLOGY Country or region before: China Applicant before: NINGXIA EPPEN BIOTECH Co.,Ltd. |
|
| TA01 | Transfer of patent application right |