WO2022124670A1 - 쉬와넬라 아틀란티카 유래 단백질을 발현하는 미생물, 및 이를 이용한 l-아미노산 생산 방법 - Google Patents
쉬와넬라 아틀란티카 유래 단백질을 발현하는 미생물, 및 이를 이용한 l-아미노산 생산 방법 Download PDFInfo
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- WO2022124670A1 WO2022124670A1 PCT/KR2021/017793 KR2021017793W WO2022124670A1 WO 2022124670 A1 WO2022124670 A1 WO 2022124670A1 KR 2021017793 W KR2021017793 W KR 2021017793W WO 2022124670 A1 WO2022124670 A1 WO 2022124670A1
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- microorganism
- amino acid
- protein
- lysine
- amino acids
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- 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
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 101150035025 lysC gene Proteins 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 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
- 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
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Definitions
- the original function may be a lysine excretion protein function (in the case of an amino acid sequence), or a function encoding a protein having the lysine excretion protein function (in the case of a nucleic acid sequence), and the desired function is a microorganism of L-amino acids (eg, L-lysine, L-arginine, or a combination thereof) may refer to a function of increasing or imparting excretion and/or production capacity.
- L-amino acids eg, L-lysine, L-arginine, or a combination thereof
- RNA-guided endonuclease system RNA-guided endonuclease system or CRISPR system
- RNA-guided endonuclease system or CRISPR system
- an RNA-guided endonuclease eg, Cas9 protein, etc.
- its coding gene or a vector comprising the gene
- a guide RNA eg, single guide RNA (sgRNA), etc.
- sgRNA single guide RNA
- a mixture eg, a mixture of an RNA-guided endonuclease protein and a guide RNA, etc.
- a complex eg, a ribonucleic acid fusion protein (RNP), a recombinant vector (eg, , an RNA-guided endonuclease-encoding gene and a vector including a guide RNA-encoding DNA, etc.)
- RNP ribonucleic acid fusion protein
- recombinant vector
- Another example is a method of increasing the L-amino acid excretion and / or production capacity of the microorganism, comprising introducing (transformation) into the microorganism the foreign protein, its encoding polynucleotide, or a recombinant vector containing the polynucleotide Or it provides a method for imparting L- amino acid excretion and / or production ability to the microorganism.
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Abstract
Description
No. | 균 주 | 단백질 등록 번호 | 게놈 등록 번호 |
1 | Shewanella oneidensis MR-1 | WP_011072781.1 | NC_004347.2 |
2 | Corynebacterium glutamicum ATCC13032 | WP_003854734.1 | NC_003450.3 |
3 | Aeromonas allosaccharophila | WP_042063605.1 | NZ_CDCB01000123 |
4 | Bacillus pseudofirmus OF4 | WP_012958260.1 | NC_013791.2 |
5 | Bacillus halodurans C-125 | WP_010899078.1 | NC_002570.2 |
6 | Natrialba aegyptia | WP_006666837.1 | NZ_AOIP01000039.1 |
7 | Haloquadratum walsbyi | WP_011571763.1 | NC_008212.1 |
8 | Thermotoga maritima MSB8 | WP_004083139.1 | NC_000853.1 |
9 | Pyrodictium delaneyi | WP_055407773.1 | NZ_CP013011.1 |
10 | Bacteroides coprophilus | WP_008140089.1 | NZ_EQ973631.1 |
11 | Shewanella atlantica HAW-EB5 | WP_126504868.1 | NZ_RXNV01000002.1 |
No. | 명칭 | DNA 서열 |
서열번호 3 | Sat-F | TAGAGGAGACACAACATGCAGGCATTTATTCAGGG |
서열번호 4 | Sat-R | TAGATGTCGGGCCCATTACAAGTAAGGCCATAACAA |
서열번호 5 | PgapA-F | AATGAGTTCCTCGAGAAACGACCGAGCCTATTGGG |
서열번호 6 | PgapA-R | TAAATGCCTGCATGTTGTGTCTCCTCTAAAGATTG |
서열번호 7 | pDZTn-1 | ACGACGCTGGTATTTCTCCC |
서열번호 8 | pDZTn-2 | TGATTGTCGATATGACCGGG |
단계 | 온도 | 시간 |
Initialization | 95 ℃ | 2분 |
Denaturation | 95 ℃ | 20초 |
Annealing | 62 ℃ | 40초 |
Elongation | 72 ℃ | 0.5~1분 |
Post Elongation | 72 ℃ | 5분 |
No. | 명칭 | DNA 서열 |
서열번호 9 | Tn-1 | TAAGGCACCGCAGAATGTAG |
서열번호 10 | Tn-2 | TAGGACTCACCATCACCGGC |
균 주 | OD562 | 소모당 (g/L) | 라이신 농도 (g/L) | 아르기닌 농도 (mg/L) |
KCCM11016P | 51 | 50 | 12.1 | 24.2 |
KCCM11016P::PgapA-Sat | 40.5 | 50 | 13.2 | 26.8 |
균 주 | OD562 | 소모당 (g/L) | 라이신 생산량 (g/L) | 아르기닌 농도 (mg/L) |
KCCM10770P | 70.3 | 50 | 8.3 | 16.6 |
KCCM10770P:: PgapA-Sat | 58.8 | 50 | 9.8 | 20.1 |
균 주 | OD562 | 소모당 (g/L) | 라이신 생산량 (g/L) | 아르기닌 농도 (mg/L) |
CJ3P | 75.5 | 50 | 3.4 | 53.4 |
CJ3P::PgapA-Sat | 69.7 | 50 | 4.5 | 78.2 |
Claims (10)
- 서열번호 1의 쉬와넬라 아틀란티카(Shewanella atlantica) 유래 막단백질 또는 이와 75% 이상의 서열 상동성을 갖는 막단백질을 발현하는, 코리네박테리움 속 미생물.
- 제1항에 있어서, 상기 코리네박테리움 속 미생물은 상기 막단백질을 암호화하는 폴리뉴클레오타이드가 도입된 것인, 코리네박테리움 속 미생물.
- 제2항에 있어서, 상기 폴리뉴클레오타이드는 서열번호 2 또는 이와 75% 이상의 서열 상동성을 갖는 핵산 서열로 표현되는 것인, 코리네박테리움 속 미생물.
- 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 막단백질을 발현하지 않는 코리네박테리움 속 미생물과 비교하여, L-아미노산의 생상능이 증가된, 코리네박테리움 속 미생물.
- 제4항에 있어서, 상기 L-아미노산은 L-라이신 또는 L-아르기닌인, 미생물.
- 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 코리네박테리움 속 미생물은 코리네박테리움 글루타미쿰인, 코리네박테리움 속 미생물.
- 제1항 내지 제3항 중 어느 한 항의 코리네박테리움 속 미생물을 포함하는, L-아미노산 생산용 조성물.
- 제7항에 있어서, 상기 L-아미노산은 L-라이신 또는 L-아르기닌인, 조성물.
- 제1항 내지 제3항 중 어느 한 항의 코리네박테리움 속 미생물을 배지에서 배양하는 단계, 및상기 배양된 미생물, 배지, 또는 이들 모두로부터 L-아미노산을 회수하는 단계를 포함하는, L-아미노산의 생산 방법.
- 제9항에 있어서, 상기 L-아미노산은 L-라이신 또는 L-아르기닌인, 방법.
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JP2022554350A JP7447294B2 (ja) | 2020-12-09 | 2021-11-29 | シュワネラ・アトランティカ由来蛋白質を発現する微生物、およびこれを利用したl-アミノ酸生産方法 |
AU2021394840A AU2021394840B2 (en) | 2020-12-09 | 2021-11-29 | Shewanella atlantica-derived protein-expressing microorganism and L-amino acid production method using same |
EP21903716.5A EP4261286A1 (en) | 2020-12-09 | 2021-11-29 | Shewanella atlantica-derived protein-expressing microorganism and l-amino acid production method using same |
CN202180016206.1A CN115605599A (zh) | 2020-12-09 | 2021-11-29 | 表达大西洋希瓦氏菌衍生的蛋白质的微生物和使用其生产l-氨基酸的方法 |
MX2023000785A MX2023000785A (es) | 2020-12-09 | 2021-11-29 | Microorganismo que expresa una proteina derivada de shewanella atlantica y metodo para producir l-aminoacidos usando el mismo. |
ZA2022/10974A ZA202210974B (en) | 2020-12-09 | 2022-10-06 | Shewanella atlantica-derived protein-expressing microorganism and l-amino acid production method using same |
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KR19990076708A (ko) * | 1995-12-22 | 1999-10-15 | 아달베르트 베. 플라텐타이히, 하 | 엑스포트 캐리어의 활성 증대를 통한 미생물사용 아미노산 제조방법 |
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KR100924065B1 (ko) | 2006-09-15 | 2009-10-27 | 씨제이제일제당 (주) | L-라이신 생산능이 향상된 코리네박테리아 및 그를 이용한 l-라이신 생산 방법 |
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MX2023000785A (es) | 2023-02-14 |
KR20220081825A (ko) | 2022-06-16 |
CN115605599A (zh) | 2023-01-13 |
JP2023517591A (ja) | 2023-04-26 |
JP7447294B2 (ja) | 2024-03-11 |
AR124292A1 (es) | 2023-03-15 |
ZA202210974B (en) | 2024-02-28 |
AU2021394840A1 (en) | 2022-08-04 |
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