KR20000067653A - Genetically engineered Escherichia coli strains and the methods to produce optically pure D- or L-lactate production using the strains - Google Patents
Genetically engineered Escherichia coli strains and the methods to produce optically pure D- or L-lactate production using the strains Download PDFInfo
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- KR20000067653A KR20000067653A KR1019990015654A KR19990015654A KR20000067653A KR 20000067653 A KR20000067653 A KR 20000067653A KR 1019990015654 A KR1019990015654 A KR 1019990015654A KR 19990015654 A KR19990015654 A KR 19990015654A KR 20000067653 A KR20000067653 A KR 20000067653A
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- lactic acid
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- 241000588724 Escherichia coli Species 0.000 title claims abstract description 90
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 82
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 title claims description 56
- 238000000034 method Methods 0.000 title description 19
- 229940116871 l-lactate Drugs 0.000 title 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 150
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 119
- 239000004310 lactic acid Substances 0.000 claims abstract description 59
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 59
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000001384 succinic acid Substances 0.000 claims abstract description 25
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000001301 oxygen Substances 0.000 claims abstract description 11
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 11
- 239000013612 plasmid Substances 0.000 claims abstract description 9
- 229930182843 D-Lactic acid Natural products 0.000 claims description 44
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 claims description 44
- 229940022769 d- lactic acid Drugs 0.000 claims description 43
- 238000012258 culturing Methods 0.000 claims description 3
- 230000037361 pathway Effects 0.000 abstract description 22
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- 210000004027 cell Anatomy 0.000 description 31
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- 238000002360 preparation method Methods 0.000 description 19
- 239000006166 lysate Substances 0.000 description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 17
- 239000008103 glucose Substances 0.000 description 17
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- 229930182823 kanamycin A Natural products 0.000 description 13
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- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 6
- 101100398785 Streptococcus agalactiae serotype V (strain ATCC BAA-611 / 2603 V/R) ldhD gene Proteins 0.000 description 6
- 101100386830 Zymomonas mobilis subsp. mobilis (strain ATCC 31821 / ZM4 / CP4) ddh gene Proteins 0.000 description 6
- 101150026107 ldh1 gene Proteins 0.000 description 6
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- 108700023175 Phosphate acetyltransferases Proteins 0.000 description 5
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- 150000003522 tetracyclines Chemical class 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 4
- 238000012742 biochemical analysis Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 238000004186 food analysis Methods 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- 108010001539 D-lactate dehydrogenase Proteins 0.000 description 3
- 102100023319 Dihydrolipoyl dehydrogenase, mitochondrial Human genes 0.000 description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- 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 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101001130148 Escherichia coli (strain K12) D-lactate dehydrogenase Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000000789 acetogenic effect Effects 0.000 description 2
- 229960000723 ampicillin Drugs 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
- 230000015572 biosynthetic process Effects 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000012269 metabolic engineering Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
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- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 229940076788 pyruvate Drugs 0.000 description 2
- 210000003370 receptor cell Anatomy 0.000 description 2
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- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UWTATZPHSA-M (R)-lactate Chemical compound C[C@@H](O)C([O-])=O JVTAAEKCZFNVCJ-UWTATZPHSA-M 0.000 description 1
- 108010092060 Acetate kinase Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241001640117 Callaeum Species 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108091000041 Phosphoenolpyruvate Carboxylase Proteins 0.000 description 1
- 101100194362 Schizosaccharomyces pombe (strain 972 / ATCC 24843) res1 gene Proteins 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009603 aerobic growth Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000000976 ink Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 1
- -1 phosphoenolpyruvate carbohydrate Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
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- 239000012137 tryptone Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B64—AIRCRAFT; AVIATION; COSMONAUTICS
- B64D—EQUIPMENT FOR FITTING IN OR TO AIRCRAFT; FLIGHT SUITS; PARACHUTES; ARRANGEMENT OR MOUNTING OF POWER PLANTS OR PROPULSION TRANSMISSIONS IN AIRCRAFT
- B64D17/00—Parachutes
- B64D17/02—Canopy arrangement or construction
- B64D17/025—Canopy arrangement or construction for gliding chutes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B64—AIRCRAFT; AVIATION; COSMONAUTICS
- B64D—EQUIPMENT FOR FITTING IN OR TO AIRCRAFT; FLIGHT SUITS; PARACHUTES; ARRANGEMENT OR MOUNTING OF POWER PLANTS OR PROPULSION TRANSMISSIONS IN AIRCRAFT
- B64D17/00—Parachutes
- B64D17/02—Canopy arrangement or construction
- B64D17/04—Canopy arrangement or construction formed with two or more canopies arranged about a common axis
Landscapes
- Engineering & Computer Science (AREA)
- Aviation & Aerospace Engineering (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
본 발명은 대장균 변이주 및 형질전환 대장균(Escherichia coli) 및 이를 이용한 D형 또는 L형 젖산의 순수 생산방법에 관한 것으로서, 더욱 상세하게는 유전공학 기술을 이용하여 대사경로를 변형시키므로 초산 및 숙신산 생성경로를 유전적으로 결손시켜 D형 젖산만을 선택적으로 생산할 수 있는 신균주 대장균 JP203, 또는 L형 젖산만을 선택적으로 생산할 수 있는 신균주 대장균 JP204을 얻고, 이 균주들을 호기적 조건에서 1차 생장시킨 후, 젖산 생산단계에서는 2차적으로 배양조건을 혐기적 조건으로 전환시켜 배양함으로써, 보다 효율적으로 그리고 보다 높은 수율로 D형 또는 L형 젖산만을 선택적으로 대량 생산할 수 있는 방법에 관한 것이다.The present invention relates to E. coli mutant strains and transformed Escherichia coli (Escherichia coli) and the pure production method of D- or L-lactic acid using the same, more specifically, because the metabolic pathway is modified using genetic engineering techniques, the production route of acetic acid and succinic acid E. coli JP203, which can selectively produce only D-type lactic acid, or Escherichia coli JP204, which can selectively produce only L-type lactic acid, is obtained by genetic defection, and these strains are grown first in aerobic conditions. In the production stage, the method is capable of selectively mass-producing only D- or L-type lactic acid in a more efficient and higher yield by culturing by converting the culture conditions into anaerobic conditions.
젖산은 식품분야, 의약분야, 화장품분야 등 산업분야에서 그 요구성이 크게 증가하고 있다. 이중 식품분야에서는 청량음료, 제과, 제빵 및 청주 제조용으로 사용되고, 의약분야에서는 칼슘 및 철분의 강화제 및 장내 소독용 등으로 사용되며, 산업적으로는 젖산 유도체가 농약 합성의 중간유도체로 사용되거나 페인트, 잉크 등에 첨가하여 사용되는 등 산업 전반에 걸쳐서 광법위하게 이용되고 있다(Vickroy, 1985, Comprehensive Biotechnology Vol. 3, 1st edition, 761-776).Lactic acid is greatly increasing in demand in industries such as food, medicine and cosmetics. In the food field, it is used for the manufacture of soft drinks, confectionery, bakery, and sake. In the pharmaceutical field, it is used as an intensifier for calcium and iron and for intestinal disinfection.In the industrial field, lactic acid derivatives are used as intermediates for the synthesis of pesticides, paints and inks. It is widely used throughout the industry, such as used in addition to (Vickroy, 1985, Comprehensive Biotechnology Vol. 3, 1st edition, 761-776).
이러한 상기 젖산의 종래의 생산방법으로는 화학 합성법과 직접발효법이 있다.Conventional production methods for such lactic acid include chemical synthesis and direct fermentation.
젖산은 그 광학적 성질에 따라 D형과 L형으로 나뉘는데, 화학 합성법은 D형과 L형이 혼합된 형태로 생산되는 방법이고, 직접발효법은 젖산균을 이용하며 젖산균의 성질에 따라 D형, L형 또는 혼합형이 생산되는 방법이다. 여기서, 상기 화학 합성법에서는 D형과 L형의 젖산이 혼합된 형태로 생산되어 젖산을 이용한 중합체나 유도체의 생산에 불리한 단점이 있다. 따라서, 이러한 단점과 함께 최근들어 직접발효법이 환경친화적인 이유로 더욱 선호되고 있는 실정이다.Lactic acid is divided into D type and L type according to its optical properties. The chemical synthesis method is produced by mixing D type and L type. The direct fermentation method uses lactic acid bacteria and D type and L type according to the properties of lactic acid bacteria. Or a mixed form is produced. Here, the chemical synthesis method is produced in the form of a mixture of D- and L-type lactic acid has a disadvantage in the production of polymers or derivatives using lactic acid. Therefore, in addition to these drawbacks, the direct fermentation method is more preferred in recent years due to environmentally friendly.
그러나, 젖산균을 이용한 직접발효법에서는 젖산균 자체의 생육이 늦고 그 영양 요구성이 복잡하여 거의 모든 젖산균이 비타민 B를 요구하고, 또한 많은 종류의 아미노산을 증식인자로 요구하는 단점이 있다(Stanier 등, 1986, The Microbial World, 5th edition, 496-501, Prentice-Hall, USA).However, in the direct fermentation method using lactic acid bacteria, the growth of lactic acid bacteria itself is complicated and its nutritional requirements are complicated, so almost all lactic acid bacteria require vitamin B and many kinds of amino acids as growth factors (Stanier et al., 1986). , The Microbial World, 5th edition, 496-501, Prentice-Hall, USA).
이에, 본 발명자들은 젖산균들을 이용한 직접발효법의 단점을 극복하고자 연구 노력한 결과, 유전공학 기술을 이용하여 대사경로를 변형시키므로 초산 및 숙신산 생성경로를 유전적으로 결손시켜 D형 또는 L형 젖산만을 선택적으로 생산할 수 있는 신균주 대장균을 얻을 수 있었다. 따라서, 본 발명은 유전공학 기술을 이용하여 대사경로를 변형시키므로 초산 및 숙신산의 생성경로를 유전적으로 결손시켜 D형 젖산만을 선택적으로 생산할 수 있는 신균주 대장균 JP203, 또는 L형 젖산만을 선택적으로 생산할 수 있는 신균주 대장균 JP204를 얻고, 이 균주들을 호기적 조건에서 1차 생장시킨 후, 젖산 생산단계에서는 2차적으로 배양조건을 혐기적 조건으로 전환시켜 배양함으로써, 보다 효율적으로 그리고 보다 높은 수율로 D형 또는 L형 젖산을 선택적으로 생산할 수 있는 방법을 제공하는 것에 그 목적이 있다.Therefore, the present inventors have tried to overcome the shortcomings of the direct fermentation method using lactic acid bacteria, and as a result of modifying the metabolic pathway by using genetic engineering technology to genetically delete acetic acid and succinic acid production pathway to selectively produce only D or L-lactic acid Escherichia coli could be obtained. Therefore, the present invention modifies the metabolic pathway using genetic engineering techniques, so that the production paths of acetic acid and succinic acid can be selectively deleted to produce only E. coli JP203 or L-lactic acid, which can selectively produce only D-type lactic acid. Escherichia coli JP204 was obtained, and these strains were grown first in aerobic conditions, and then, in the lactic acid production stage, secondary cultures were converted to anaerobic conditions to form D more efficiently and with higher yields. Another object is to provide a method for selectively producing L-lactic acid.
도 1은 대장균 RR1 pta::Tnpho'-3을 이용하여 혐기적 조건에서의 고농도 D형 젖산 생산 결과를 나타낸 그래프이고,1 is a graph showing the results of production of high concentration D-lactic acid in anaerobic conditions using E. coli RR1 pta :: Tnpho'-3,
도 2는 대장균 RR1 pta::Tnpho'-3을 이용하여 산소제한 조건에서의 고농도 D형 젖산 생산 결과를 나타낸 그래프이고,2 is a graph showing the results of the production of high concentration D-lactic acid under oxygen-limiting conditions using E. coli RR1 pta :: Tnpho'-3,
도 3은 본 발명에 따라 제조한 신균주 대장균 JP203을 이용하여 혐기적 조건에서의 고농도 D형 젖산 생산을 나타낸 그래프이며,3 is a graph showing the production of high concentration D-lactic acid in anaerobic conditions using the E. coli JP203 prepared according to the present invention,
도 4는 본 발명에 따라 제조한 신균주 대장균 JP204을 이용하여 혐기적 조건에서의 고농도 L형 젖산 생산을 나타낸 그래프이다.Figure 4 is a graph showing the production of high concentration L-lactic acid in anaerobic conditions using the new strain E. coli JP204 prepared according to the present invention.
본 발명은 초산 및 숙신산 생산능이 결핍되고 D형 젖산에 대한 선택적 생산능을 갖는 신규 변이주 대장균 JP203(KCTC 0599BP)과 플라스미드 pLS65를 함유하고 L형 젖산에 대한 선택적 생산능을 갖는 신규 변이주 대장균 JP204(KCTC 0600BP)를 그 특징으로 한다.The present invention is a novel mutant strain E. coli JP203 (KCTC 0599BP) and plasmid pLS65 which lacks acetic acid and succinic acid production capacity and has a selective production capacity for type D lactic acid and has a selective production capacity for L type lactic acid. 0600BP).
또한, 본 발명은 상기 대장균 JP203(KCTC 0599BP)과 대장균 JP204(KCTC 0600BP) 각각을 호기적 조건으로 배양한 후, 혐기적 또는 산소 제한조건에서 배양하여 D형 젖산 또는 L형 젖산만을 선택적으로 생산하는 방법을 포함한다.In addition, the present invention, after culturing each of the Escherichia coli JP203 (KCTC 0599BP) and Escherichia coli JP204 (KCTC 0600BP) in aerobic conditions, and incubated under anaerobic or oxygen restriction conditions to selectively produce only D-type lactic acid or L-type lactic acid It includes a method.
이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.
본 발명은 신규 변이주 대장균 JP203(KCTC 0599BP)과 대장균 JP204(KCTC 0600BP) 및 이를 이용하여 보다 효율적으로 젖산을 선택적으로 대량 생산하는 방법에 관한 것이다.The present invention relates to a novel mutant strain E. coli JP203 (KCTC 0599BP) and E. coli JP204 (KCTC 0600BP) and a method for mass production of lactic acid more efficiently using the same.
본 발명은 형질전환된 대장균을 이용하는 방법에 관한 것으로, 우선 본 발명과 관련된 대장균의 일반적인 특성은 영양 요구성이 간단하고, 약 20분의 생장주기를 갖고 있어 생육이 빠르고, 최적온도 37℃, pH 7.0, 그람-음성의 종속영양생물로서, 이용할 수 있는 기질 또한 다양하여 발효에 응용하기 쉽다는 것이다(Bergey's Manual of Systematic Bacteriology, Vol.1, 414 ∼ 417, Williams ＆ Wilkins, USA). 또한, 대장균은 호기성 성장은 물론, 상기한 바와 같이 혐기성 성장도 가능케 하는 대사잠재력을 갖는 통기성 미생물이다.The present invention relates to a method of using transformed Escherichia coli. First, the general characteristics of Escherichia coli according to the present invention have a simple nutritional requirement and a growth cycle of about 20 minutes, so that the growth is fast, and the optimum temperature is 37 ° C., pH. 7.0, Gram-negative heterotrophs, are also available in a variety of substrates for ease of fermentation (Bergey's Manual of Systematic Bacteriology, Vol. 1, 414-417, Williams & Wilkins, USA). In addition, Escherichia coli is a breathable microorganism having metabolic potential which enables not only aerobic growth but also anaerobic growth as described above.
상기와 같은 유용한 대사잠재력과 함께, 대장균은 생리학적 및 유전학적 연구가 가장 많이 되어 있는 미생물이고, 이에 따른 대사조절이 용이하고 대사공학의 결과를 쉽게 예측할 수 있으며, 또한 유전공학적 조작에 따른 재조합 균주의 생산 및 대사공학 기술을 아주 쉽게 적용할 수 있는 장점이 있다.In addition to the above useful metabolic potential, E. coli is a microorganism with the most physiological and genetic studies, and accordingly, it is easy to control metabolism and easily predict the results of metabolic engineering. It is very easy to apply the technology of production and metabolic engineering.
[D형 젖산에 대한 선택적 생산능을 갖는 대장균 및 이를 이용한 D형 젖산의 대량생산 방법][E. coli having selective production capacity for D-lactic acid and mass production method of D-lactic acid using the same]
본 발명은 D형 젖산을 생산할 수 있는 신규 대장균 변이주 및 이를 이용한 D형 젖산의 생산방법을 제공하는 바, 이를 상세히 하기로 한다.The present invention provides a novel E. coli mutant strain capable of producing D-type lactic acid and a production method of D-type lactic acid using the same, which will be described in detail.
초산, 젖산, 포름산, 알코올, 숙신산 등을 생산하는 혼합발효형(mixed acid fermentation) 장내 세균인 대장균을 이용하여 D형 젖산만을 선택적으로 생산하려면 해당과정을 통해 생성된 피루브산 농도를 높이거나, 배양액의 pH를 낮춰 D형 젖산 탈수소화 효소의 활성을 높여야 한다[Clark, 1989, FEMS Microbiol. Rev., 63:223-234]. 그러나, 상기한 경우에도 다른 유기산과의 혼합형으로 나오므로 대장균의 발효경로중 초산경로를 차단하여 세포내 피루브산 농도를 상대적으로 증가시키고 혐기적 조건이나 산소 제한상태로 배양하여 세포내로 유입된 탄소원의 대사흐름을 D형 젖산쪽으로 유도해야 한다.To selectively produce only D-lactic acid using Escherichia coli, a mixed acid fermentation intestine that produces acetic acid, lactic acid, formic acid, alcohol, succinic acid, increase the concentration of pyruvic acid produced by the relevant process, The pH should be lowered to increase the activity of type D lactate dehydrogenase [Clark, 1989, FEMS Microbiol. Rev., 63: 223-234. However, even in the above case, since it comes out as a mixed form with other organic acids, it blocks the acetic acid route in the fermentation route of E. coli, and relatively increases the intracellular pyruvate concentration and incubates in anaerobic or oxygen-restricted state to metabolize the carbon source introduced into the cell. The flow should be directed to type D lactic acid.
이에, 본 발명자들은 초산 생산능만이 결핍되고 D형 젖산에 대한 선택적 생산능을 갖는 변이주 대장균 RR1 pta::Tnpho'-1(KCTC 0095BP)와 이를 이용한 D형 젖산의 순수 생산방법을 이미 대한민국 특허 제122428호를 통해 제시한 바 있다.Thus, the present inventors have already patented the Korean strain of strain E. coli RR1 pta :: Tnpho'-1 (KCTC 0095BP) having only acetic acid production capacity and selective production capacity for D-type lactic acid and a pure production method of D-type lactic acid using the same. No. 122428.
초산경로의 차단은 초산 합성에 필요한 효소 포스포트랜스아세틸라아제(phosphoacetyltransferase, pta)와 아세테이트 키나아제(acetate kinase, ack) 중에서 첫 번째 효소를 암호화 하는 유전자에 가나마이신(kanamycin) 표식단편 DNA가 삽입되어 그 활성이 유전학적으로 제거된 균주에 P1 파이지를 감염시켜 용균액을 얻고 이를 원하는 균주에 감염시키는 P1 파아지를 이용한 형질도입법[Silhavy 등, 1984, Experiments with gene fusions, Cold Spring Harbor Laboratory, N.Y., USA]으로 원하는 균주의 염색체의 포스포트랜스아세틸라아제를 암호화하는 유전자에 가나마이신 표식단편 DNA를 삽입하여 효소의 활성을 유전학적으로 제거함으로써 이루어진다.Blocking of the acetic acid pathway involves the insertion of kanamycin marker fragment DNA into the gene encoding the first of the enzymes, phosphoacetyltransferase (pta) and acetate kinase (ac,), which are required for acetic acid synthesis. Transfection using P1 phage that infects P1 phage to genetically eliminated activity to obtain lysate and infects the desired strain [Silhavy et al., 1984, Experiments with gene fusions, Cold Spring Harbor Laboratory, NY, USA] by inserting the kanamycin marker fragment DNA into the gene encoding the phosphotransacetylase of the chromosome of the desired strain by genetically removing the activity of the enzyme.
구체적으로는, 대장균 OW1(KCTC 2308)으로부터 pta::TnphoA'-3 유전형을 갖도록 제조한 표식유전자를 갖는 공여세포 대장균 OW1 pta::TnphoA'-3(한국과학기술원 미생물유전학실)[신 등, 1995, Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR, J. Bacteriol., 177: 4696-4702]을 배지에서 배양한 후, 배양액에 P1 파아지를 접종하여 용균액을 얻는다. 이렇게 얻은 P1 용균액은 정상 바이러스와 형질도입 바이러스 입자를 갖게 된다.Specifically, E. coli OW1 pta :: TnphoA'-3 (Microbial Genetics Laboratory, Korea Advanced Institute of Science and Technology) having a marker gene prepared to have the pta :: TnphoA'-3 genotype from Escherichia coli OW1 (KCTC 2308) [Shin et al., 1995, Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR, J. Bacteriol., 177: 4696-4702], followed by incubation of P1 phage in culture to obtain a lysate. The P1 lysate thus obtained had normal virus and transduced virus particles.
수용세포 대장균을 배지에서 배양한 후, 원심분리하여 세포를 분리하고 상기와 같이 얻은 P1 용균액을 접종시켜 수용세포를 P1 파아지로 감염시켰다. 감염시킨 세포를 가나마이신이 들어있는 배지에 도말한 후 생성된 세포 군락으로부터 초산생산 결핍균을 선별한다. 본 발명의 대장균을 제조하기 위한 수용세포 대장균으로는 대장균 RR1(KCTC 1437), EJ500(한국과학기술원 미생물 유전학실), HB101(KCTC 1467)등이 이용될 수 있으며, 그 중 대장균 RR1이 바람직하다.Recipient cells were cultured in E. coli, and then centrifuged to separate the cells and inoculated with the obtained P1 lysate to infect the recipient cells with P1 phage. The infected cells are plated in a medium containing kanamycin and selected for acetic acid deficiency bacteria from the cell population. E. coli RES1 (KCTC 1437), EJ500 (Microbial Genetics Laboratory, Korea Advanced Institute of Science and Technology), HB101 (KCTC 1467) and the like may be used as the recipient cell E. coli for the production of E. coli, and among them, E. coli RR1 is preferred.
위에서 제조된 초산 생산 결핍 대장균을 이용하여 종균 배양 및 주배양을 실시하되, 처음에는 호기적 조건에서 다음에는 혐기적 조건 또는 산소제한 조건으로 전환하여 발효를 수행한다.The spawn culture and main culture are carried out using the acetic acid-deficient E. coli prepared above, but the fermentation is carried out by first switching from aerobic conditions to anaerobic or oxygen-restricted conditions.
호기적 조건으로 배양할 때, 생산균은 초산 생산이 유전학적으로 결핍되었으므로 호기적 조건에서의 주 대사산물인 초산생산이 거의 없고, 포름산과 에탄올 또한 축적하지 않는다. 적합한 세포농도가 되는 시기에 배양액 중에 질소가스를 불어넣어 완전한 혐기적 조건으로 전환시키거나, 교반 속도 및 통기 속도를 낮추어 산소 제한 조건으로 전환시켜 배양을 계속한다. 산소제한 조건에서 교반 속도는 100 ∼ 500 rpm의 범위 내일 수 있고, 200 rpm정도가 바람직하며, 통기속도는 0.1 ∼ 0.5 ℓ/분, 바람직하기로는 0.3 ℓ/분으로 한다. 상기 발효들에서는, 발효중간에 부족한 탄소원을 보충하기 위해 배지내 포도당 농도가 8 ∼ 12 g/ℓ가 될 때마다 포도당 용액을 첨가하며, 일정 시간 후, 유입된 탄소원에 대해 70% 이상의 수율로 D형 젖산을 고농도로 배양액 중에 축적한다.When cultured in aerobic conditions, the producing bacteria are genetically deficient in acetic acid production, so there is almost no acetic acid production, which is the main metabolite in aerobic conditions, and also does not accumulate formic acid and ethanol. Nitrogen gas is blown into the culture at a suitable cell concentration to convert to complete anaerobic conditions, or the agitation and aeration rates are reduced to oxygen-limiting conditions to continue the culture. Under the oxygen limitation conditions, the stirring speed may be in the range of 100 to 500 rpm, about 200 rpm is preferred, and the aeration rate is 0.1 to 0.5 L / min, preferably 0.3 L / min. In the fermentations, a glucose solution is added every time the glucose concentration in the medium becomes 8-12 g / l to compensate for the insufficient carbon source during the fermentation, and after a certain time, in a yield of 70% or more with respect to the introduced carbon source, Type lactic acid accumulates in the culture at high concentrations.
그러나, 상기와 같이 초산경로를 차단하여 D형 젖산을 생산할 수 있도록 제조된 대장균을 D형 젖산의 생산에 이용할 경우에 탄소원의 5 ∼ 15%가 숙신산으로 배지 내에 축적되어 D형 젖산의 수율이 떨어지는 문제가 있다.However, when using E. coli prepared to block the acetic acid pathway to produce D-type lactic acid for production of D-type lactic acid, 5-15% of the carbon source accumulates in the medium as succinic acid, resulting in a poor yield of D-type lactic acid. there is a problem.
이에, 숙신산의 생성을 차단하고 D형 젖산의 수율을 높이기 위하여 초산 경로와 숙신산 경로가 동시에 차단된 균주를 제조하여 순수 D형 젖산 생산 균주로 육종하였는 바, 이를 본 발명의 특징으로 한다. 그리고, 이를 상세히 하면 다음과 같다.Thus, in order to block the production of succinic acid and to increase the yield of D-type lactic acid, the strain was prepared at the same time the acetic acid path and succinic acid path was bred and bred as pure D-type lactic acid producing strain, which is a feature of the present invention. And, in detail this is as follows.
우선, 초산경로의 차단은 상기한 방법 즉, 대한민국 특허 제122428호에 개시된 초산경로 차단 방법과 같다.First, the blocking of the acetate route is the same as the above method, that is, the acetate route blocking method disclosed in Korean Patent No. 122428.
한편, 숙신산 경로의 차단은 숙신산 경로의 첫 분기점에 해당하는 포스포에놀피루브산 카르복실라아제(phosphoenolpyruvate carboxylase)를 암호화하는 유전자에 클로람페니콜(chloramphenicol) 표식단편 DNA를 P1 파아지를 이용한 형질도입법으로 삽입하여 효소의 활성을 유전학적으로 제거함으로써 이루어진다.On the other hand, the blocking of the succinic acid pathway was inserted by transduction method using chloramphenicol marker fragment DNA into the gene encoding phosphoenolpyruvate carboxylase corresponding to the first branch of the succinic acid pathway. By genetically removing the activity of the enzyme.
구체적으로는, 대장균 W3110(ATCC27325)으로부터 ppc::Cmr유전형을 갖도록 제조된 표식유전자를 갖는 공여세포 대장균 KJE103(생명공학연구소 발효시스템 R.U.)[장 등, 1999, Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1, Appl. Environ. Microbiol., 65:1384-1389]으로부터 D형 젖산 생산균주의 제조시에 사용된 것과 같은 방법으로 숙신산 경로 차단을 위한 P1 용균액을 얻는다. D형 젖산 생산 균주의 제조시와 같은 방법으로 제조된 초산 경로가 차단된 D형 젖산 생산 균주에 위에서 제조된 숙신산 경로 차단을 위한 P1 용균액을 감염시키고 감염시킨 세포를 가나마이신과 클로람페니콜이 들어있는 LB 평판배지에 도말한 후 생성된 세포군락으로부터 순수 초산 및 숙신산 생산 결핍 균주를 선별한다.Specifically, donor cell E. coli KJE103 (Bio Fermentation System RU), which has a marker gene prepared to have a ppc :: Cm r genotype from Escherichia coli W3110 (ATCC27325) [Chang et al., 1999, Homofermentative production of D- or L- lactate in metabolically engineered Escherichia coli RR1, Appl. Environ. Microbiol., 65: 1384-1389, to obtain a P1 lysate for blocking the succinic acid pathway in the same manner as was used in the preparation of the D-lactic acid producing strain. Type D lactic acid-producing strains blocked by the acetic acid pathway prepared in the same manner as in the production of type D-lactic acid-producing strains were infected with P1 lysate for blocking the succinic acid pathway prepared above, and the cells containing kanamycin and chloramphenicol were contained. Pure acetic acid and succinic acid deficient strains are selected from the resulting cell populations after plating on LB plate medium.
본 발명에서 제조된, RR1 pta::Tnpho'-3 ppc::cat의 유전형을 가져 초산 및 숙신산 생산능이 결핍되고 D형 젖산에 대한 선택적 생산능을 갖는 신규 변이주 대장균 JP203은 99년 4월 14일자로 한국과학기술원 생명공학연구소 유전자은행(KCTC)에 기탁번호 KCTC 0599BP로서 기탁되었다.The new mutant strain E. coli JP203 prepared in the present invention having a genotype of RR1 pta :: Tnpho'-3 ppc :: cat, which lacks acetic acid and succinic acid production and has a selective production capacity for type D lactic acid, was dated April 14, 99. It was deposited with KCTC 0599BP at the Korea Advanced Institute of Science and Technology.
위에서 제조된 초산 및 숙신산 생산 결핍 균주를 이용하여 상기 초산 생산능만이 결핍되고 D형 젖산에 대한 선택적 생산능을 갖는 신규 변이주 대장균을 이용한 D형 젖산 생산의 경우와 동일한 배지와 방법으로 발효를 수행하면 배지내에 숙신산의 축적이 없이 90% 이상의 수율로 순수 D형 젖산을 고농도로 축적할 수 있다.Fermentation was carried out using the same medium and method as in the case of production of D-type lactic acid using a novel mutant strain E. coli, which lacks only acetic acid-producing ability and has a selective production capacity for D-type lactic acid, using the acetic acid and succinic acid-deficient strains prepared above. In this case, pure D-lactic acid can be accumulated at a high concentration in a yield of 90% or more without accumulation of succinic acid in the medium.
[L형 젖산에 대한 선택적 생산능을 갖는 대장균 및 이를 이용한 L형 젖산의 대량생산 방법][E. coli having selective production capacity for L-lactic acid and mass production method of L-lactic acid using the same]
본 발명은 L형 젖산을 생산할 수 있는 신규 대장균 변이주 및 이를 이용한 L형 젖산의 생산방법을 또한 제공하는 바, 이를 상세히 하기로 한다.The present invention also provides a novel Escherichia coli mutant strain capable of producing L-type lactic acid and a production method of L-type lactic acid using the same, which will be described in detail.
대장균은 L형 젖산 생산 경로에 관여하는 L형 젖산 탈수소효소(L-lactate dehydrogenase)를 갖고 있지 않으므로 발효법에 의하여 L형 젖산을 생산하려면 외래의 젖산 탈수소화 효소를 도입하여 발현시켜야 한다. 즉, 본 발명에서는 Lactobacillus casei(ATCC393) 에서 유래된 L형 젖산 탈수소 효소를 암호화 하는 유전자를 갖고 있는 플라스미드 pLS65(한국과학기술원 생명과학과)[김 등, 1991, Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene, Appl. Environ. Microbiol., 57: 2413-2417]를 대장균에 도입하여 이 효소를 발현시킴으로서 L형 젖산을 생산하는 균주를 제조하였다. 이 경우, D형 젖산을 생산하는 균주의 제조에서와 같이 대장균의 발효 경로 중 초산 경로를 차단하여 세포내의 피루브산 농도가 상대적으로 높아진 균주에 다시 대장균의 D형 젖산의 생성 경로를 차단함으로써 혐기적 조건이나 산소제한 조건에서 세포내로 유입된 탄소원의 흐름을 외래의 L형 젖산 탈수소화 효소를 통한 L형 젖산쪽으로 유도한다. 초산경로의 차단은 포스포트랜스아세틸라아제를 암호화 하는 유전자를 테트라사이클린(tetracyclin) 표식단편 DNA를 P1 파아지를 이용한 형질도입법으로 삽입하여 포스포트랜스아세틸라아제의 활성을 유전적으로 제거함으로써 이루어진다. D형 젖산 경로의 차단을 위하여는 초산경로가 차단된 균주의 D형 젖산 탈수소화 효소를 암호화하는 유전자에 가나마이신 표식단편 DNA를 P1파아지를 이용한 형질도입법[Silhavy 등, 1984, Experiments with gene fusions, Cold Spring Harbor Laboratory, N.Y., USA]으로 삽입하여 D형 젖산 탈수소화 효소의 활성을 유전적으로 제거하였다. 초산 경로와 D형 젖산 경로가 모두 차단된 균주에 Lactobacillus casei에서 유래된 L형 젖산 탈수소 효소를 암호화 하는 유전자를 갖고 있는 플라스미드 pLS65를 도입하여 L형 젖산만을 선택적으로 생산하는 균주를 제조하였다.E. coli does not have L-lactate dehydrogenase, which is involved in the L-lactic acid production pathway. Therefore, in order to produce L-lactic acid by fermentation, an exogenous lactic acid dehydrogenase must be introduced and expressed. That is, in the present invention, plasmid pLS65 (Dept. of Biological Sciences, Korea Advanced Institute of Science and Technology) having a gene encoding L-type lactate dehydrogenase derived from Lactobacillus casei (ATCC393) [Kim et al., 1991, Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene, Appl. Environ. Microbiol., 57: 2413-2417] was introduced into E. coli to express this enzyme, producing a strain producing L-lactic acid. In this case, as in the production of strains producing D-type lactic acid, the acetic acid pathway is blocked in the fermentation pathway of E. coli, thereby blocking the production path of E-type D-lactic acid in Escherichia coli again to a strain having a relatively high concentration of pyruvate in cells. Under the condition of oxygen restriction, the flow of carbon source into the cell is directed to L-lactic acid through foreign L-lactic acid dehydrogenase. The blocking of the acetic acid pathway is achieved by genetically eliminating the activity of phosphotransacetylase by inserting a gene encoding phosphotransacetylase into the tetracycline-labeled fragment DNA by transduction using P1 phage. In order to block the D-lactic acid pathway, transfection using P1 phage with kanamycin-labeled fragment DNA into a gene encoding the D-type lactate dehydrogenase of a strain blocked with acetic acid [Silhavy et al., 1984, Experiments with gene fusions] , Cold Spring Harbor Laboratory, NY, USA] to genetically remove the activity of type D lactate dehydrogenase. A strain that selectively produces only L-lactic acid was introduced by introducing plasmid pLS65 having a gene encoding L-lactic acid dehydrogenase derived from Lactobacillus casei into a strain that blocked both the acetic acid pathway and the D-type lactic acid pathway.
구체적으로는 대장균 OW1로부터 Φ(pta-1::Tn10-lacZ) 유전형을 갖도록 제조한 표식유전자를 갖는 공여세포 대장균 OW1 Φ(pta-1::Tn10-lacZ)(한국과학기술원 미생물 유전학실)[신 등, 1995, Modulation of flagellar expression in Escherichia coli by actyl phosphate and the osmoregulator OmpR, J. Bacteriol., 177: 4696-4702]로부터 D형 젖산 생산균주의 제조시에 사용된 것과 같은 방법으로 초산경로 차단을 위한 P1 용균액을 얻는다. 또한, 대장균 LCB320으로부터 ldhA::Kmr유전형을 갖도록 제조한 표식유전자를 갖는 공여세포 대장균 NZN117(ldhA::Kmr)[Bunch 등, 1997, The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli, Microbiology, 143:187-195]로부터 D형 젖산 생산균주의 제조시와 같은 방법으로 D형 젖산 경로 차단을 위한 P1 용균액을 얻는다.Specifically, donor cell E. coli OW1 Φ (pta-1 :: Tn10-lacZ) having a marker gene prepared to have a Φ (pta-1 :: Tn10-lacZ) genotype from E. coli OW1 (Microbial Genetics Laboratory, Korea Advanced Institute of Science and Technology) [ Shin, et al., 1995, Modulation of flagellar expression in Escherichia coli by actyl phosphate and the osmoregulator OmpR, J. Bacteriol., 177: 4696-4702]. Obtain P1 lysate. In addition, donor cell E. coli NZN117 (ldhA :: Km r ) having a marker gene prepared from the E. coli LCB320 to have a ldhA :: Km r genotype [Bunch et al., 1997, The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli, Microbiology, 143: 187-195] to obtain the P1 lysate for blocking the D-lactic acid pathway in the same manner as in the production of the D-lactic acid producing strain.
먼저, D형 젖산 생산균주의 제조시와 같은 방법으로 D형 젖산 경로 차단을 위한 P1 용균액을 수용세포 대장균에 감염시키고 감염시킨 세포를 가나마이신이 들어있는 LB 평판배지에 도말한 후 생성된 세포군락으로부터 D형 젖산 경로 결핍 균주를 선별한다. 이 균주에 같은 방법으로 초산경로 차단을 위한 P1 용균액을 감염시키고, 감염시킨 세포를 가나마이신과 테트라사이클린이 들어있는 LB 평판배지에 도말한 후 생성된 세포군락으로부터 초산 및 D형 젖산 생산 결핍 균주를 선별한다.First, the P1 lysate for blocking the D-lactic acid pathway is infected with E. coli, and the infected cells are plated on LB plate medium containing kanamycin in the same manner as in the production of the D-type lactic acid-producing strain. Type D lactic acid pathway deficient strains are selected from the colonies. The strain was infected with P1 lysate for blocking the acetic acid pathway in the same manner, and the infected cells were plated on LB plate medium containing kanamycin and tetracycline, and then deficient in acetic acid and D-lactic acid production from the cell population. Screening.
위에서 얻어진 초산 및 D형 젖산 생산 결핍 균주에 CaCl2방법으로 Lactobacillus casei에서 유래된 L형 젖산 탈수소 효소를 암호화하는 유전자를 갖고 있는 플라스미드 pLS65를 도입하고 가나마이신, 테트라사이클린 및 암피실린을 함유하는 LB 평판배지에 도말하여 형질전환된 균주를 선별한다.LB plate medium containing kanamycin, tetracycline and ampicillin was introduced into the acetic acid and D-lactic acid-deficient strains obtained above by introducing the plasmid pLS65 carrying the gene encoding the L-lactic acid dehydrogenase derived from Lactobacillus casei by CaCl 2 method. The transformed strains are selected by plating.
본 발명에서 제조된, RR1 Φ(pta-1::Tn10-lacZ) ldhA::Kmr유전형을 가지며 플라스미드 pLS65를 함유하여 초산 및 숙신산 생산능이 결핍되고 L형 젖산에 대한 선택적 생산능을 갖는 신규 변이주 대장균 JP204(pLS65)은 99년 4월 14일 자로 한국과학기술원 생명공학연구소 유전자은행(KCTC)에 기탁번호 KCTC 0600BP로서 기탁되었다.Novel mutants having the RR1 Φ (pta-1 :: Tn10-lacZ) ldhA :: Km r genotype produced in the present invention, containing plasmid pLS65, lacking acetic acid and succinic acid production, and having selective production for L-lactic acid Escherichia coli JP204 (pLS65) was deposited on April 14, 99 with the accession number KCTC 0600BP to KCTC.
위에서 제조된 균주를 L형 젖산 생산 대장균 균주를 이용하여 상기한 D형 젖산 생산의 경우와 동일한 배지와 방법으로 발효를 수행하면 L형 젖산을 고농도로 축적할 수 있다.L-lactic acid can be accumulated at a high concentration by fermenting the strain prepared above using L-lactic acid-producing E. coli strains in the same medium and method as in the case of D-type lactic acid production.
이하, 본 발명을 하기의 실시예에 의해 보다 상세히 설명하겠는 바, 본 발명이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.
제조예 1 : 초산 생산능이 결핍되고 D형 젖산에 대한 선택적 생산능을 갖는 대장균의 제조Preparation Example 1: Preparation of Escherichia coli lacking acetic acid production capacity and selective production capacity for D-lactic acid
대장균 OW1으로부터 pta::TnphoA'-3 유전형을 갖도록 제조된 대장균 OW1 pta::TnphoA'-3(한국과학기술원 미생물 유전학실)을 2 ㎖ TGC배지(0.1% 포도당 및 10mM 염화칼슘을 포함하는 박토-트립톤 액체배지(Bacto-Trypton broth)에 37℃에서 하룻밤 동안 배양한 배양액을 다시 3㎖ TGC배지에 1% 접종하여 37℃에서 1시간 정도 재배양하였다. 600 ㎚에서의 흡광도가 약 0.15가 되면 P1 파아지(1010 pfu/㎖)를 50㎖ 첨가하여 2 ∼ 3시간 방치하였다. 배양액이 맑아지면 0.1 ㎖ 클로로포름을 넣고 혼합하여 3000 x g에서 10분 동안 원심분리한 후, 상등액을 취해 형질도입 P1 용균액으로 사용하였다.Escherichia coli OW1 pta :: TnphoA'-3 (Korea Advanced Institute of Science and Technology microbial genetics) prepared to have a pta :: TnphoA'-3 genotype from Escherichia coli OW1 was subjected to bacterium-trip containing 2 ml TGC medium (0.1% glucose and 10 mM calcium chloride). The culture medium incubated overnight at 37 ° C. in a ton liquid medium (Bacto-Trypton broth) was inoculated again in 1 ml of 3 ml TGC medium and incubated for 1 hour at 37 ° C. When the absorbance at 600 nm reached about 0.15, P1 50 ml of phage (1010 pfu / ml) was added and left for 2 to 3 hours.When the culture was clear, 0.1 ml of chloroform was added, mixed and centrifuged at 3000 xg for 10 minutes, and then the supernatant was taken as a transduced P1 lysate. Used.
수용세포 대장균 RR1(KCTC 1437)은 LB배지((박토-트립톤(Bacto-Tryptone) 10 g/ℓ, 박토-효모엑기스(Bacto-yeast extract) 5g/ℓ, 소금(NaCl) 5g/ℓ, pH=7.2)에서 하루밤 배양한 후, 3000 x g에서 10분간 원심분리하여 침전된 세포만을 취하고 배양액 부피의 절반정도 되는 10 mM MgSO4와 5 mM CuCl2용액을 첨가하여 혼탁시켰다.Receptor cell E. coli RR1 (KCTC 1437) was treated with LB medium ((10 g / l Bacto-Tryptone, 5g / l Bacto-yeast extract, 5g / l salt (NaCl), pH). = 7.2) overnight, followed by centrifugation at 3000 xg for 10 minutes to collect only the precipitated cells and turbidity by adding 10 mM MgSO 4 and 5 mM CuCl 2 solutions, which are half the volume of the culture.
4개의 튜브에 수용세포 RR1 현탁액을 0.1㎖씩 넣은 후, 여기에 공여세포 대장균 OW1 pta::TnphoA'-3로부터 얻은 P1 용균액을 각각 0 ㎖, 10 ㎖, 50 ㎖, 100 ㎖씩 넣고 혼합하여 상온에서 20분 동안 방치했다. 상기 혼합물에 1 M 구연산나트륨을 100 ㎖넣고 12000 rpm에서 30초 동안 원심분리하여 침전물을 취한 후, 1 M 구연산나트륨 100 ㎖에 재현탁시켰다. 다시 12000 rpm에서 30초 동안 원심분리하여 LB배지 0.9 ㎖와 100 mM 구연산나트륨 0.1 ㎖를 첨가하여 37℃에서 한시간동안 방치한 후, 가나마이신이 들어 있는 평판배지에 도말하여 37℃에서 배양하였다. 초산생성 결핍 변이주의 증식속도 차이에 의해 작은 군락 크기의 대장균을 초산 생산 결핍 변이주(RR1 pta::TnphoA'-3)로서 선택하였다.0.1 ml of the receptive cell RR1 suspension was added to each of the four tubes, followed by mixing 0 ml, 10 ml, 50 ml and 100 ml of the P1 lysate from the donor cell E. coli OW1 pta :: TnphoA'-3. It was left at room temperature for 20 minutes. 100 ml of 1 M sodium citrate was added to the mixture, and the precipitate was taken by centrifugation at 12000 rpm for 30 seconds, and then resuspended in 100 ml of 1 M sodium citrate. After centrifugation at 12000 rpm for 30 seconds, 0.9 ml of LB medium and 0.1 ml of 100 mM sodium citrate were added and left at 37 ° C. for one hour, and then plated on a plate medium containing kanamycin and incubated at 37 ° C. Small colony-sized E. coli was selected as the acetogenic deficiency mutant strain (RR1 pta :: TnphoA'-3) due to the difference in proliferation rate of the acetogenic deficiency mutants.
제조예 2 : 초산 및 숙신산 생산능이 결핍되고 D형 젖산에 대한 선택적 생산능을 갖는 신규 변이주 대장균의 제조Preparation Example 2 Preparation of a Novel Mutant Escherichia Coli Having Deficient Acetic Acid and Succinic Acid Production Capacity and Selective Production Capacity for Type D Lactic Acid
대장균 KJE 103(ppc::Cmr)(생명공학연구소 발효시스템 R.U.) 균주로부터 상기 제조예 1에서와 같은 방법으로 포스포에놀피루브산 카르복실라아제를 유전적으로 불활성화시키기 위한 형질도입 P1 용균액을 얻었다.Transgenic P1 lysate for genetically inactivating phosphoenolpyruvic acid carboxylase in the same manner as in Preparation Example 1 above from E. coli KJE 103 (ppc :: Cm r ) (Bioferment System RU) Got.
상기 제조예 1에서 제조된 초산 생산 결핍 변이주(RR1 pta::TnphoA'-3)를 수용세포로 사용하기 위하여 제조예 1에서와 같은 방법으로 LB배지에서 배양하고 위에서 제조된 포스포에놀피루브산 카르복실라아제를 유전적으로 불활성화 시키기 위한 형질도입 P1 용균액을 같은 방법으로 처리한 다음, 가나마이신과 클로람페니콜이 들어있는 LB 평판배지에 도말하여 배양한 후, 성장하는 대장균을 초산 및 숙신산 생산 결핍 변이주(RR1 pta::TnphoA'-3 ppc::Cmr)로서 선택하였다. 초산 생산 결핍 변이주의 증식속도 차이와 탄소원으로 포도당만을 함유하는 최소배지에서의 숙신산 생산 결핍균주의 숙신산 요구성에 의해 초산 및 숙신산 생산 결핍 변이주를 최종적으로 선택하였다.In order to use the acetic acid-producing deficiency strain (RR1 pta :: TnphoA'-3) prepared in Preparation Example 1 as a recipient cell, the phosphoenolpyruvate carbohydrate was prepared in the same manner as in Preparation Example 1 and prepared above. The transgenic P1 lysate for genetically inactivating the carboxylase was treated in the same manner, and then plated and cultured in LB plate medium containing kanamycin and chloramphenicol, and the growing Escherichia coli was deficient in acetic acid and succinic acid production. (RR1 pta :: TnphoA'-3 ppc :: Cm r ). Variants of acetic acid and succinic acid production deficiency were finally selected by the difference in the growth rate of acetic acid production deficiency strains and the succinic acid requirement of succinic acid production deficiency strains in minimal medium containing glucose as a carbon source.
제조예 3 : 초산 및 D형 젖산 생산능이 결핍되고 L형 젖산에 대한 선택적 생산능을 갖는 신규 변이주 대장균의 제조Preparation Example 3: Preparation of a new mutant strain E. coli lacking acetic acid and D-type lactic acid production capacity and having selective production capacity for L-type lactic acid
상기 제조예 1과 동일한 방법으로, 대장균 OW1으로부터 Φ(pta-1::Tn10-lacZ)의 유전형을 갖도록 제조한 대장균 OW1 Φ(pta-1::Tn10-lacZ) 구(한국과학기술원 미생물 유전학실)[신 등, 1995, Modulation of flagellar expression in Escherichia coli by actyl phosphate and the osmoregulator OmpR, J. Bacteriol., 177: 4696-4702]로부터 상기 제조예 1에서와 같은 방법으로 포스포트랜스아세틸라아제를 유전적으로 불활성화 시키기 위한 형질도입 P1 용균액을 얻었다.In the same manner as in Preparation Example 1, E. coli OW1 Φ (pta-1 :: Tn10-lacZ) sphere prepared from E. coli OW1 to have a genotype of Φ (pta-1 :: Tn10-lacZ) (Microbial Genetics Laboratory, Korea Advanced Institute of Science and Technology) Phosphotransacetylase was synthesized in the same manner as in Preparation Example 1 from [Shin et al., 1995, Modulation of flagellar expression in Escherichia coli by actyl phosphate and the osmoregulator OmpR, J. Bacteriol., 177: 4696-4702]. Transduced P1 lysate was obtained for genetic inactivation.
또한, 상기 형질도입 P1 용균액의 수득방법과 동일하게 대장균 NZN 117 (ldhA::Kmr)(Southern Illinois University at Carbondale, Department of Mocrobiology)[Bunch 등, 1997, The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli, Microbiology, 143:187-195] 균주로부터 D형 젖산 탈수소화 효소를 유전적으로 불활성화시키기 위한 형질도입 P1 용균액을 얻었다.In addition, E. coli NZN 117 (ldhA :: Km r ) (Southern Illinois University at Carbondale, Department of Mocrobiology) [Bunch et al., 1997, The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli, Microbiology, 143: 187-195] transgenic P1 lysate for genetically inactivating type D lactate dehydrogenase was obtained.
수용세포 대장균 RR1을 실시예 1에서와 같은 방법으로 LB배양지에서 배양하고 위에서 제조된 D형 젖산 탈수소화 효소를 유전적으로 불활성화 시키기 위한 형질도입 P1 용균액을 상기 제조예 1과 동일한 방법으로 처리하여 가나마이신이 들어있는 LB 평판배지에 도말하여 배양한 다음, 성장하는 대장균을 D형 젖산 생산 결핍 변이주( RR1 ldhA::Kmr)로서 선택하였다. 이 D형 젖산 생산 결핍 변이주에 상기에서 제조된 포스포트랜스아세틸라아제를 유전적으로 불활성화시키기 위한 형질도입 P1 용균액을 같은 방법으로 처리하고 가나마이신과 테트라사이클린이 함유된 LB 평판배지에 도말하여 배양하였다. 초산 생산 결핍 변이주의 증식속도 차이에 의해 작은 군락크기의 대장균을 초산 및 D형 젖산생산 결핍 변이주를 선택하였다.Receptor cell Escherichia coli RR1 was cultured in LB culture in the same manner as in Example 1 and transduced P1 lysate for genetically inactivating the D-lactic acid dehydrogenase prepared above was treated in the same manner as in Preparation Example 1 above. Staining and incubating in LB plate medium containing kanamycin, the growing E. coli was selected as a type D lactate deficient mutant (RR1 ldhA :: Km r ). The transgenic P1 lysate for genetically inactivating the phosphotransacetylase prepared above was treated in the same manner as the D-lactic acid deficient mutant strain and plated on LB plate medium containing kanamycin and tetracycline. Incubated. Variants of acetic acid-deficient mutants were selected as acetic acid and D-type lactate deficient mutants.
이 초산 및 D형 젖산생산 결핍 변이주를 LB배지에 접종하여 37℃에서 하룻밤 동안 배양하고 홈이 있는 500 ㎖ 플라스크에 100 ㎖의 LB 배지를 넣고 하룻방 키운 배양액 0.5 ㎖를 접종한 후 37℃의 진탕배양기에서 호기적으로 배양하다가 600 ㎚에서의 흡광도가 약 0.4가 되면 얼음에 10분간 놓아두었다. 배양액을 50 ㎖ 원심분리용 튜브에 옮겨 5000 x g에서 10 분간 원심분리하여 상등액은 버리고 세포 침전물에 미리 차게 해둔 멸균된 CaCl2용액을 원래의 1/5 부피가 되게 가하여 세포를 혼탁시킨 다음, 얼음위에 15분간 놓아두었다가 5000 x g로 4℃에서 10분 동안 원심분리하였다. 상등액은 버리고 차게 해둔 멸균된 CaCl2용액을 원래의 1/5 부피가 되게 가하여 세포를 혼탁시킨 다음 30 분간 얼음위에 놓아 두었다가 1.5 ㎖ 미니원심분리용 튜브에 나누어 놓았다.The acetic acid and type D lactate deficient mutants were inoculated in LB medium and incubated overnight at 37 ° C., inoculated with 100 ml of LB medium in a grooved 500 ml flask and inoculated with 0.5 ml of the broth grown overnight, followed by shaking at 37 ° C. After aerobic incubation in the incubator, the absorbance at 600 nm was about 0.4 and left on ice for 10 minutes. Transfer the culture to a 50 ml centrifuge tube and centrifuge at 5000 xg for 10 minutes to discard the supernatant and add 1/5 volume of sterile CaCl 2 solution pre-cold to the cell precipitate to turbid the cells, and then place them on ice. Leave for 15 minutes and centrifuge at 5000 xg for 10 minutes at 4 ° C. The supernatant was discarded and the sterilized CaCl 2 solution was added to a volume of 1/5 of the original, the cells were clouded, left on ice for 30 minutes and then divided into 1.5 ml minicentrifuge tubes.
위에서 처리한 세포현탁액 0.2 ㎖에 Lactobacillus casei(ATCC393)에서 유래된 L형 젖산 탈수소 효소를 암호화 하는 유전자를 갖고 있는 플라스미드 pLS65 DNA(한국과학기술원 생명과학과)[김 등, 1991, Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene, Appl. Environ. Microbiol., 57: 2413-2417]를 10㎕ 가하여 섞은 다음 얼음위에 30분간 놓아 두었다가 42℃ 수온조에서 90초간 열처리한 다음 얼음물에 옮겨 1 ∼ 2분간 차게 식힌 후, LB배지를 0.8 ㎖ 가하여 37℃에서 60분간 흔들지 않고 배양하였다. 이 세포액을 가나마이신, 테트라사이클린 그리고 암피실린이 함유된 LB 평판배지에 도말하여 성장하는 세포의 군락을 Lactobacillus casei에서 유래된 L형 젖산 탈수소 효소를 암호화 하는 유전자를 갖고 있는 플라스미드 pLS65를 가지는 초산 및 D형 젖산생산 결핍 변이주(RR1 pta::TnphoA'-1 ldhA::Kmr(pLS65))로 최종적으로 선택하였다.Plasmid pLS65 DNA (Dept. of Biological Science and Technology, Korea Advanced Institute of Science and Technology) containing a gene encoding L-lactic acid dehydrogenase derived from Lactobacillus casei (ATCC393) in 0.2 ml of the cell suspension treated above [Kim et al., 1991, Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene, Appl. Environ. Microbiol., 57: 2413-2417] was added thereto, mixed, and left on ice for 30 minutes, heat-treated in a 42 ° C. water bath for 90 seconds, cooled in ice water for 1 to 2 minutes, and cooled to 37 ° C. by adding 0.8 ml of LB medium. Incubated at 60 minutes without shaking. The cell solution was plated on LB plate medium containing kanamycin, tetracycline and ampicillin to grow a colony of cells. Acetic acid and type D with plasmid pLS65 containing a gene encoding L-type lactate dehydrogenase derived from Lactobacillus casei. It was finally selected as the lactic acid production deficient mutant strain (RR1 pta :: TnphoA'-1 ldhA :: Km r (pLS65)).
실시예 1 : 혐기조건에서 초산 생산능이 결핍된 대장균을 이용한 D형 젖산의 생산방법Example 1 Production Method of Type D Lactic Acid Using Escherichia Coli Lacking Acetic Acid Production Capacity in Anaerobic Conditions
상기 제조예 1에서 제조된 초산 생산능이 결핍된 대장균을 이용하여 다음 표 1 에 나타낸 배지성분을 이용하여 종균배양과 주배양을 실시하였다.Using E. coli lacking the acetic acid production capacity prepared in Preparation Example 1 was performed spawn culture and main culture using the media components shown in Table 1 below.
홈이 있는 500 ㎖ 플라스크에 100 ㎖의 상기 표 1의 배지를 넣고, 상기 제조예 1에서 제조된 초산 생성능이 결핍된 대장균을 접종한 후, 37℃의 진탕배양기에서 호기적 조건에서 하룻밤 동안 종균배양하여 배양기에 최종부피의 3 %가 되도록 접종하였다.100 ml of the medium of Table 1 was placed in a 500 ml flask with a groove, inoculated with E. coli deficient in acetic acid production ability prepared in Preparation Example 1, and then incubated overnight in aerobic conditions at 37 ° C. in an aerobic condition. The inoculator was inoculated to 3% of the final volume.
본 배양에서 배양기는 5 리터 발효배양기에서 배양액의 부피를 3 리터로 하고 배양온도 37℃, pH는 7.0으로 조정하여 수행하였다. 배양액의 호기적 조건을 유지하기 위하여 교반속도는 800 ∼ 1000 rpm으로, 통기속도는 2 ∼ 3 ℓ/분으로 변화시키면서 배양하였다. 부족한 탄소원를 보충하기 위해 배양액 중 포도당의 농도가 10g/ℓ 이하가 되면 70%의 농축된 포도당액 200 ㎖을 첨가하였다. 배양액 중 세포농도가 10 g/ℓ가 되었을 때, 배양기에 질소가스를 불어넣어 완전한 혐기적 조건에서 D형 젖산의 생산을 유도하였고, 그 결과는 첨부도면 도 1에 나타내었다. 혐기적 조건으로 전환한 후 약 45시간만에 D형 젖산이 리터당 60 그램이상 배양액에 축적되었다. 수율로는 소비된 포도당 1g당 0.68g의 D형 젖산 이 생산되었다. 그리고, D형 젖산을 D형 젖산에 특이적인 D형 젖산 탈수소 효소에 의한 효소적 방법(Boehringer Mannheim Co.의 생화학 및 식품 분석 Kit 사용)으로 확인하였다.In this culture, the incubator was performed by adjusting the volume of the culture solution to 3 liters in a 5 liter fermentation incubator and adjusting the culture temperature to 37 ° C. and pH to 7.0. In order to maintain the aerobic conditions of the culture solution, the culture rate was changed while changing the agitation speed to 800 to 1000 rpm and the aeration rate to 2-3 l / min. In order to make up for the insufficient carbon source, when the concentration of glucose in the culture medium was 10 g / l or less, 200 ml of 70% concentrated glucose solution was added. When the cell concentration in the culture medium was 10 g / ℓ, nitrogen gas was blown into the incubator to induce the production of type D lactic acid under complete anaerobic conditions, and the results are shown in FIG. About 45 hours after the transition to anaerobic conditions, D-lactic acid accumulates in more than 60 grams of culture per liter. In yield, 0.68 g of D-lactic acid was produced per 1 g of glucose consumed. In addition, type D lactic acid was identified by an enzymatic method (using a biochemical and food analysis kit of Boehringer Mannheim Co.) by the type D lactic acid dehydrogenase specific to type D lactic acid.
실시예 2 : 산소제한 조건에서 초산 생산능이 결핍된 대장균을 이용한 D형 젖산의 생산방법Example 2 production method of D-type lactic acid using E. coli lacking acetic acid production capacity under oxygen limit conditions
상기 실시예 1과 동일한 배지 및 균주을 이용, 동일 조건으로 호기적 배양을 수행하다가 배양액 중 세포농도가 10 g/ℓ가 되었을 때 교반속도를 200 rpm으로, 통기속도를 0.3ℓ/분으로 낮추어 산소제한 조건으로 전환시켰다. 상기 제조예 2와 마찬가지로 부족한 탄소원의 보충을 위해 배양액중 포도당의 농도가 10 g/ℓ 이하가 되면 70%의 농축된 포도당액 200 ㎖을 첨가하였다. 산소제한 조건에서는 산소제한이 시작된 후, 60시간 만에 리터당 60g 이상의 D형 젖산이 배양액 내에 축적되었다. 수율로는 소비된 포도당 1 g당 0.62g의 D형 젖산이 생산되었다. 그리고, D형 젖산을 D형 젖산에 특이적인 D형 젖산 탈수소효소에 의한 효소적 방법(Boehringer Mannheim Co.의 생화학 및 식품 분석 Kit 사용)으로 확인하였다.Using the same medium and strain as in Example 1, the aerobic culture was carried out under the same conditions. When the cell concentration in the culture solution reached 10 g / l, the agitation rate was reduced to 200 rpm and the aeration rate was reduced to 0.3 l / min. Condition was converted. As in Preparation Example 2, 200 ml of concentrated glucose solution of 70% was added when the concentration of glucose in the culture medium was 10 g / l or less to supplement the insufficient carbon source. Under the oxygen limit condition, more than 60 g of D-type lactic acid per liter accumulated in the culture medium within 60 hours after the oxygen limit was started. In yield, 0.62 g of Form D lactic acid was produced per 1 g of glucose consumed. In addition, type D lactic acid was identified by an enzymatic method (using a biochemical and food analysis kit of Boehringer Mannheim Co.) by the type D lactic acid dehydrogenase specific to type D lactic acid.
실시예 3 : 혐기조건에서의 초산 및 숙신산 생산능이 결핍된 신규 변이주 대장균을 이용한 D형 젖산의 생산방법Example 3 Production Method of Type D Lactic Acid Using Novel Mutant Escherichia Coli Lacking Acetic Acid and Succinic Acid Production Capacity in Anaerobic Conditions
상기 제조예 2에서 제조된 초산 및 숙신산 생산능이 결핍된 변이주(RR1 pta::TnphoA'-3 ppc::Cmr)를 상기 제조예 2와 동일 배지, 동일조건으로 호기적 배양을 수행하다가 배양액중 세포농도가 10 g/ℓ가 되었을 때 배양기에 질소가스를 불어넣어 완전한 혐기적 조건에서 D형 젖산의 생산을 유도하였고, 실시예 1과 마찬가지로 부족한 탄소원의 보충을 위해 배양액중 포도당의 농도가 10 g/ℓ 이하가 되면 70%의 농축된 포도당액 200 ㎖을 첨가하였다. 혐기조건으로 전환한 후 48 시간 만에 리터당 60 g 이상의 D형 젖산이 배양액 내에 축적되었다. 수율로는 소비된 포도당 1g당 0.90g의 D형 젖산 이 생산되어 초산경로만 결핍된 젖산 생산 균주에 의한 D형 젖산 생산의 경우(소비된 포도당 1 g당 0.6 ∼ 0.7 g의 D형 젖산의 생산수율)에 비하여 수율이 크게 향상되었다. 그리고, D형 젖산을 D형 젖산에 특이적인 D형 젖산 탈수소효소에 의한 효소적 방법(Boehringer Mannheim Co. 의 생화학 및 식품 분석 Kit 사용)으로 확인하였다.The mutant strain (RR1 pta :: TnphoA'-3 ppc :: Cm r ) lacking acetic acid and succinic acid production capacity prepared in Preparation Example 2 was subjected to aerobic culture under the same conditions and conditions as in Preparation Example 2, When the cell concentration reached 10 g / l, nitrogen gas was blown into the incubator to induce the production of type D lactic acid under complete anaerobic conditions. As in Example 1, the concentration of glucose in the culture medium was 10 g to supplement the insufficient carbon source. 200 ml of concentrated glucose solution was added when it was below / l. Within 48 hours after conversion to anaerobic conditions, more than 60 g of type D lactic acid per liter accumulated in the culture. As a yield, 0.90 g of D-lactic acid is produced per 1 g of glucose consumed, and the production of D-lactic acid by lactic acid-producing strains lacking only the acetic acid pathway (production of 0.6-0.7 g of D-lactic acid per 1 g of glucose consumed Yield greatly improved compared to the yield). In addition, D-type lactic acid was identified by an enzymatic method (using a biochemical and food analysis kit of Boehringer Mannheim Co.) by D-type lactic acid dehydrogenase specific to D-type lactic acid.
실시예 4 : 산소제한 조건에서의 초산 및 D형 젖산 생산능이 결핍된 신규 변이주 대장균을 이용한 L형 젖산의 생산방법Example 4 Production of L-Lactic Acid Using New Mutant Escherichia Coli Lacking Acetic Acid and D-Lactate Production Capacity Under Oxygen Restriction Conditions
상기 제조예 3에서 제조한 L형 젖산 생산균주를 상기한 실시예 3와 동일한 배지에서 동일한 조건으로 호기적 배양을 수행하다가 배양액 중 세포농도가 10 g/ℓ가 되었을때, 교반속도를 200 rpm으로, 통기속도를 0.3 ℓ/분으로 낮추어 산소제한 조건으로 전환시켰다. 부족한 탄소원의 보충을 위해 배양액 중 포도당의 농도가 10 g/ℓ 이하가 되면 70%의 농축된 포도당액 200 ㎖을 첨가하였다. 산소제한 조건에서 산소제한이 시작된 후 60시간 만에 리터당 45 g 이상의 L형 젖산이 배양액 내에 축적되었다. 수율로는 소비된 포도당 1 g당 0.61g의 L형 젖산이 생산되었다. 그리고, L형 젖산을 L형 젖산에 특이적인 L형 젖산 탈수소효소에 의한 효소적 방법(Boehringer Mannheim Co. 의 생화학 및 식품 분석 Kit 사용)으로 확인하였다.When the L-type lactic acid producing strain prepared in Preparation Example 3 was subjected to aerobic incubation under the same conditions in the same medium as in Example 3 above, and the cell concentration in the culture medium was 10 g / l, the stirring speed was 200 rpm. , The aeration rate was lowered to 0.3 L / min to switch to oxygen-limiting conditions. In order to supplement the insufficient carbon source, when the concentration of glucose in the culture medium was 10 g / l or less, 200 ml of 70% concentrated glucose solution was added. More than 45 g of L-lactic acid per liter accumulates in the culture 60 hours after the start of oxygen restriction under oxygen restriction conditions. In yield, 0.61 g of L-lactic acid was produced per 1 g of glucose consumed. In addition, L-lactic acid was identified by an enzymatic method (using a biochemical and food analysis kit of Boehringer Mannheim Co.) by L-lactic acid dehydrogenase specific to L-lactic acid.
상술한 바와 같이, 본 발명은 유전공학 기술을 이용하여 대사경로를 변형시키므로 초산 및 숙신산 생성경로를 유전적으로 결손시켜 D형 젖산만을 선택적으로 생산할 수 있는 신균주 대장균 JP203, 또는 L형 젖산만을 선택적으로 생산할 수 있는 신균주 대장균 JP204을 얻고, 이 균주들을 호기적 조건에서 1차 생장시킨 후, 젖산 생산단계에서는 2차적으로 배양조건을 혐기적 조건으로 전환시켜 배양함으로써, 보다 효율적으로 그리고 보다 높은 수율로 D형 또는 L형 젖산만을 선택적으로 대량 생산할 수 있는 효과가 있다.As described above, the present invention modifies the metabolic pathway using genetic engineering techniques, so that only the strain E. coli JP203, or L-lactic acid, which can selectively produce only D-type lactic acid by genetically deleting acetic acid and succinic acid production pathways. E. coli JP204 that can be produced is obtained, these strains are grown first in aerobic conditions, and then in the lactic acid production stage, the culture conditions are converted to anaerobic conditions in a second step, so as to be more efficient and with higher yield. There is an effect that can selectively mass-produce only D- or L-type lactic acid.
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| KR101540742B1 (en) * | 2013-11-27 | 2015-07-30 | 롯데케미칼 주식회사 | Method for Producing Lactic acid Using Microalgae as a Nitrogen Source |
| WO2015170914A1 (en) * | 2014-05-09 | 2015-11-12 | 씨제이제일제당 (주) | Microorganism capable of enhancing lactic acid production and method for producing lactic acid using same |
| CN114277065A (en) * | 2021-12-30 | 2022-04-05 | 万华化学集团股份有限公司 | Method for co-producing lactic acid and succinic acid through mixed fermentation |
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| KR101540742B1 (en) * | 2013-11-27 | 2015-07-30 | 롯데케미칼 주식회사 | Method for Producing Lactic acid Using Microalgae as a Nitrogen Source |
| WO2015170914A1 (en) * | 2014-05-09 | 2015-11-12 | 씨제이제일제당 (주) | Microorganism capable of enhancing lactic acid production and method for producing lactic acid using same |
| CN114277065A (en) * | 2021-12-30 | 2022-04-05 | 万华化学集团股份有限公司 | Method for co-producing lactic acid and succinic acid through mixed fermentation |
| CN114277065B (en) * | 2021-12-30 | 2023-10-13 | 万华化学集团股份有限公司 | Method for co-production of lactic acid and succinic acid by mixed fermentation |
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