KR940001307B1 - Microorganism for producing l-lysine - Google Patents
Microorganism for producing l-lysine Download PDFInfo
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본 발명은 L-라이신을 생산하는 신규 미생물에 관한 것으로, 좀더 구체적으로는 공지의 L-라이신 생산 변이주인 코리네박테리움 글리타미쿰 KFCC 10014를 변이처리하여 얻은, L-루이신 유사체인 4-아자루이신에 내성을 지닌 균주에 관한 것이다.The present invention relates to a novel microorganism producing L-lysine, more specifically, L- leucine analogue 4-, obtained by mutating the known L- lysine production mutant Corynebacterium glycamicum KFCC 10014 It relates to a strain resistant to azaleucine.
L-라이신은 필수아미노산의 일종으로 가축의 사료, 식품첨가제, 의약원료, 영양제 등으로 사용되고 있으며, 특히 L-라이신 함량이 부족한 곡류의 가축사료에 첨가함으로써 전체적인 아미노산 조성을 양호하게 변화시켜, 사료효율을 증가시킬 수 있다고 알려져 수요가 급증하고 있는 추세이다 이러한 L-라이신의 생산은 발효법과 효소법이 병행되어 왔으나, 유전자조작 기술 및 세포융합 기술의 응용으로 고역가 균주육종이 가능하게 됨으로써, 최근에는 발효법에 의한 생산이 우위를 확보하고 있다.L-lysine is a kind of essential amino acid, which is used as feed, food additives, pharmaceutical ingredients, and nutritional supplements of livestock. The production of L-lysine has been combined with fermentation and enzymatic methods, but it is possible to breed high titer strains by application of genetic engineering techniques and cell fusion techniques. Production has the upper hand.
한편, L-라이신 생산균주인 경우 L-루이신 영양요구성을 부여시키는 것이 공지의 기술로 되어 있는데, 이는 L-라이신 생합성 경로에 관련된 디히드로피코리네이트 신세타제(Dihydrodipicolinate synthetase)의 유전자 발현을 L-루이신이 억제하기 때문인 것으로 알려져 있다(Agric. Biol. Chem, 42(8), 1501-1506, 1978).Meanwhile, in the case of L-lysine producing strains, it is known to impart L-leucine nutrient composition, which is responsible for the gene expression of Dihydrodipicolinate synthetase related to L-lysine biosynthesis pathway. L-Leucine is known to inhibit (Agric. Biol. Chem, 42 (8), 1501-1506, 1978).
본 발명에 앞서 본 발명자들은 코리네박테리움 글루타미쿰 kFCC 10014에 L-루이신 영양요구성 등을 도입하여 획득한 변이균주 코리네박테리움 글루타미쿰 KFCC 10672를 사용하여, L-라이신 발효에서 L-루이신 첨가조건을 연구한 결과를 특허출원한 바 있다(대한민국 특허공고번호 91-4368). 그러나 유가식 발효의 현장적용에서 L-루이신 농도를 발효중에 일정하게 유지시키는 것이 매우 어려워 L-라이신 비생산속도(specific production rate)가 배양과정에서 변화하고, 배양후반에는 L-루이신의 영양요구성 때문에 균체의 생리활성이 약화되어 L-라이신의 생산성이 둔화되는 단점을 보였다. 또한 L-루이신이 비교적 고가의 아미노산이기 때문에 L-라이신의 제조원가를 상승시키는 주요인이 되며, 배양중 유가식으로 첨가시켜야함으로 발효공정을 복잡하게 만드는 원인으로 나타났다.Prior to the present invention, the present inventors used a mutant strain Corynebacterium glutamicum KFCC 10672 obtained by introducing L-leucine nutritional composition into corynebacterium glutamicum kFCC 10014, and in fermentation of L-lysine The results of studying the conditions for adding L-leucine have been patented (Korean Patent Publication No. 91-4368). However, in the field application of fed-batch fermentation, it is very difficult to keep the L-leucine concentration constant during fermentation, so the specific production rate of L-lysine changes during the cultivation process. Due to the constitution, the biological activity of the cells was weakened and the productivity of L-lysine was shown to be slow. In addition, since L- leucine is a relatively expensive amino acid, it is a major factor to increase the production cost of L- lysine, and it has been shown to cause the fermentation process to be complicated by adding to the fed-batch during the culture.
따라서, 본 발명자들은 L-루이신을 자체 생합성하면서 L-라이신 생산에 영향을 주지않는 새로운 변이 균주를 획득하여 L-라이신을 제조함으로써 종래방법의 단점을 해결하였다.Therefore, the present inventors solved the shortcomings of the conventional method by obtaining L- lysine by obtaining a new mutant strain which does not affect L-lysine production while self-synthesizing L-leucine.
본 발명을 좀더 구체적으로 설명하면 다음과 같다.The present invention will be described in more detail as follows.
L-루이신이 제외된 배지에서 성장가능한 코리넥박테리움 글루타미쿰 KFCC 10014를 0.1몰 인산 완충용액(pH 6.0)에 107-108세포/ml로 현탁시키고, 32℃에서 10분 내지 30분간 N-메틸-N'-니트로-N-니트로소구아니딘(NTG)을 처리하여 변이를 유발시켰다. 그 다음 L-루이신 유사체 10-1000mg/l의 농도가 함유된 최소평판 한천배지에 변이처리한 균체현탁액 0.1-0.3ml(필요한 경우에는 농축한다)을 도말하고, 32℃에서 3~8일간 배양하여 L-루이신 유사체 내성균조를 획득하였다.Corynebacterium glutamicum KFCC 10014, grown in L-leucine-free medium, was suspended in 0.1 mole phosphate buffer (pH 6.0) at 10 7 -10 8 cells / ml, and 10 minutes to 30 minutes at 32 ° C. Mutations were induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Next, smear 0.1-0.3 ml of mutated cell suspension (concentrate if necessary) into a minimal flat agar medium containing a concentration of 10-1000 mg / l L-leucine analogues and incubate at 32 ° C. for 3 to 8 days. L-leucine analog resistant strains were obtained.
최소배지의 조성은 다음과 같으며 영양요구성 아미노산은 200mg/l씩 첨가하여 사용하였다.The composition of the minimum medium was as follows, and nutritional constituent amino acids were used by adding 200mg / l.
최소배지조성(증류서 1리터)Minimum medium composition (1 liter of distillation)
포도당 10g, 황산암모늄 2g, KH2PO40.2g, K2HPO40.2g, MgSO4·7H2O 0.1g, 비오틴 100μg, 치아민 염산염 100μg, NaP4O7·10H2O 30μg, (NH4)6Mo7O27·H2O 40μg, ZnSO4·7H2O 10μg, CuSO4·5H2O 300μg, MnCl2·4H2O 10μg, FeCl2·6H2O 1mg, (pH 7-8)10 g of glucose, 2 g of ammonium sulfate, 0.2 g of KH 2 PO 4, 0.2 g of K 2 HPO 4 , 0.1 g of MgSO 4 · 7H 2 O, 100 μg of biotin, 100 μg of thiamin hydrochloride, 30 μg of NaP 4 O 7 · 10H 2 O, (NH 4 ) 6 Mo 7 O 27 · H 2 O 40μg, ZnSO 4 · 7H 2 O 10μg, CuSO 4 · 5H 2 O 300μg, MnCl 2 · 4H 2 O 10μg, FeCl 2 · 6H 2 O 1mg, (pH 7-8)
획득한 균주의 영양요구성 및 유사체 내성은 공지의 레프리카법에 의해 확인하거나 특정 유사체가 함유된 적당한 한천배지에 도말하여 확인하였다.Nutrient composition and analog resistance of the obtained strains were confirmed by known replica method or by plating on appropriate agar medium containing specific analogs.
변이처리하여 얻은 균주들은 호기적 조건(진탕배양 250~300rpm, 발효조의 경우 0.5~1.0vvm의 통기) 및 pH 5~8, 25~37℃의 온도조건하에서 발효배지에서 배양하여 L-라이신을 배양액에 축적시켰다. 또한 발효조에서 배양하는 경우에는 수회 추가당을 공급하는 유가식 발효법으로 L-라이신을 제조하였으며, 본 발명에 있어서 균주의 발효능력은 유가식 발효법으로 측정하였다.Strains obtained by mutating were cultured in fermentation broth under aerobic conditions (shaking culture 250-300 rpm, aeration of 0.5-1.0vvm for fermenters) and pH 5-8, 25-37 ° C. to culture L-lysine Accumulated in. In addition, L-lysine was prepared by a fed-batch fermentation method to supply several additional sugars when cultured in a fermenter, and the fermentation capacity of the strain in the present invention was measured by a fed-batch fermentation method.
발효배지의 조성은 다음과 같다.The composition of the fermentation medium is as follows.
발효배지조성(공정수 1리터)Fermentation medium composition (1 liter of process water)
포도당 75g, 황산암모늄 30g, 효모추출물 5g, KH2PO41g, MgSO4·7H2O 0.4g, FeSO4·7H2O 0.01g, MnSO4·4H2O 0.0μg, 비오틴 300μg, 티아민 염산염 5001g, 칼슘 판토넨산 0.01g(pH 6-8).Glucose 75g, Ammonium sulfate 30g, Yeast extract 5g, KH 2 PO 4 1g, MgSO 4 · 7H 2 O 0.4g, FeSO 4 · 7H 2 O 0.01g, MnSO 4 · 4H 2 O 0.0μg, Biotin 300μg, Thiamine hydrochloride 5001g , Calcium pantonenoic acid 0.01 g (pH 6-8).
배양액의 균체성장 정도는 562nm에서 흡광도(optical density)로 측정하였으며, 당분석은 공지의 버트란트법으로 시행하였다. 또한 L-라이신 정량은 공지의 산성 닌히드린법(acid-ninhydrin 법) 또는 고속액체크로마토그라피법(HPLC)에 의해 분석을 했으며, L-루이신 정량은 L-루이신 영양요구성을 지닌 페디오코커스 아시디락티시(Pediococcus acidilactici) ATCC 8042를 이용한 생물학적 분석법(Bioassay) 또는 고속액체 크로마토그라피법을 사용하여 분석하였다.The cell growth of the culture was measured by optical density at 562 nm, and the sugar analysis was performed by a known butland method. In addition, L-lysine quantification was analyzed by known acid-ninhydrin method or high-performance liquid chromatography (HPLC), and L-leucine quantification was pediose with L-leucine nutrient composition. Analysis was performed using Bioassay or Fast Liquid Chromatography using Pediococcus acidilactici ATCC 8042.
이하 실시에에서 본 발명을 구체적으로 설명하고자 한다.Hereinafter, the present invention will be described in detail.
[실시예 1]Example 1
코리네박테리움 클루타미쿰 KFCC 10014를 30L 발효조에서 배양온도 32° C, 배양 pH 6.5~7.5, 통기량 0.5~1.0vvm을 유지하면서 유가식으로 배양한 다음, 최종 배양액을 아미노산 자동분석기를 사용하여 아미노산 조성을 분석하여 표 1에 기재하였다. L-루이신 첨가량에 따른 L-라이신 생산성의 변화를 관찰하기 위하여, L-루이신 0~0.6g/l를 발효배지에 첨가하여 플라스크에서 48~72시간동안 배양하여 L-라이신 생산에 미치는 영향을 검토하고 결과를 표 2에 기재하였다.Corynebacterium glutamicum KFCC 10014 was cultivated in a 30L fermenter in a fed-batch maintaining a culture temperature of 32 ° C., culture pH 6.5-7.5, and aeration 0.5-1.0vvm. The amino acid composition is analyzed and listed in Table 1. In order to observe the change in L-lysine productivity according to the amount of L-leucine added, the effect of L-leucine 0-0.6 g / l to the fermentation medium and incubated in the flask for 48-72 hours to affect the production of L-lysine Was reviewed and the results are shown in Table 2.
[표 1]TABLE 1
표1. 코리네박테리움 클루타미쿰 KFCC 10014의 배양액내 아미노산 조성Table 1. Amino Acid Composition in Cultures of Corynebacterium glutamicum KFCC 10014
[표 2]TABLE 2
표2. L-루이신 첨가가 코리네박테리움 글루타미쿰 KFCC 10014의 L-라이신 생산에 미치는 영향Table 2. Effect of L-Leucine Addition on L-Lysine Production of Corynebacterium glutamicum KFCC 10014
표2의 결과로부터 알 수 있는 바와 같이 L-루이신 첨가량이 증가함에 따라 L-라이신의 생산이 감소됨을 알 수 있었다. 따라서 L-루이신에 의한 L-라이신의 생산억제 현상을 해제시키고 발효액중의 L-루이신 축적량을 최소화시키면, L-라이신 생산성이 향상될 것을 예상하여 실시예 2와 같은 방법으로 변이주를 얻은 다음 L-라이신의 축적량을 측정하였다.As can be seen from the results of Table 2, it was found that the production of L-lysine was reduced as the amount of L-leucine added increased. Therefore, by releasing the inhibition of production of L-lysine by L-leucine and minimizing the accumulation of L-leucine in the fermentation broth, it is expected that the productivity of L-lysine will be improved. The accumulation of L-lysine was measured.
코리네박테리움 글루타미쿰 KFCC 10014를 0.1몰 인산 완충용액(pH 6.0)에 107~108세포/ml로 현탁시킨 다음, N-메틸-N'-니트로-N-니트로소구아니딘을 최종농도가 200~500μg/ml이 되도록 첨가하고, 32℃에서 10분 내지 30분간 처리하는 방법으로 인공변이시켜, L-루이신 유사체인 4-아자루이신 0~1000mg/l와 항생제의 일종인 리팜피신이 25~50mg/l각 각각 함유된 호모세린 강화(200mg/l) 최소평판 한천배지에서 앞서 언급한 방법으로 도말후, 항온배양하며 표 3의 인공변이주를 개발하였다. 변이균주 획득과정에서 각 변이균주의 L-루이신 유사체에 대한 내성도를 조사하기 위하여 0~1000mg/l의 4-아자루이신이 함유된 최소평판 한천배지에서 배양한 결과를 표 4에 기재하였다.Suspension of Corynebacterium glutamicum KFCC 10014 in 0.1 mol phosphate buffer (pH 6.0) at 10 7-10 8 cells / ml, followed by final concentration of N-methyl-N'-nitro-N-nitrosoguanidine Is added to 200 ~ 500μg / ml, and artificially mutated by the method of treatment for 10 minutes to 30 minutes at 32 ℃, the L- leucine analogue 4-Azaleucine 0-1000mg / l and a kind of antibiotic Rifampicin Homoserine-enriched (200mg / l) minimal flat agar medium containing 25 ~ 50mg / l each was plated by the above-mentioned method, incubated and incubated in Table 3. In order to investigate the resistance to L-leucine analogs of each strain strain during the acquisition of the strain strains, the results of incubation in a minimal flat agar medium containing 4-azaleucine of 0-1000 mg / l are shown in Table 4.
[표 3]TABLE 3
표3. L-라이신 생산 변이균주 및 특성Table 3. L-lysine-producing mutant strains and their characteristics
Hse:호모세린, AEC:S-(β-아미노에틸)-L-시스테인, AL:4-아자루이신, Rif:리팜피신, -:영양요구성 또는 효소활성 없음, +:비영양요구성 또는 효소활성 있음, L:leaky 형질지님, r:내성지님.Hse: Homoserine, AEC: S- (β-aminoethyl) -L-cysteine, AL: 4-Azaleucine, Rif: Rifampicin,-: No nutritional or enzymatic activity, +: Non-nutritive or enzyme Active, L: leaky trait, r: resistant.
[표 4]TABLE 4
표4. L-라이신 생산 변이균주의 4-아자루이신 내성도Table 4. 4-Azaleucine Resistance of L-Lysine-Producing Mutant
-성장 못함, +성장함-Not growing, + growing
이들 변이균주의 L-라이신 생산능력을 조사하기 위하여, 250ml 플라스크에 발효배지 20ml와 영양요구성 아미노산을 200mg/l씩 첨가한 후, 표 4의 균주들을 1 백금이 접종하 다음, 48~72시간 배양한 결과를 표5에 기재하였다.In order to investigate the L-lysine production capacity of these mutant strains, 20 ml of fermentation medium and 200 mg / l of nutrient-containing amino acids were added to a 250 ml flask, followed by inoculation of 1 platinum by the strains of Table 4, followed by 48 to 72 hours. The culture results are shown in Table 5.
[표 5]TABLE 5
표5. 코리네박테리움 글루타미쿰 KFCC 10014과 L-루이신 유사체 내성균주의 플라스크 발효결과Table 5. Flask Fermentation Results of Corynebacterium glutamicum KFCC 10014 and L-leucine Analogue Resistance Strains
이상의 실시예에서 발효배양액 L-라이신 염산염 농도가 향상되고 L-루이신이 축적되지 않는 코리네박테리움 글루타미쿰 CS-13을 최종선별하여 1991. 11. 2.자로 사단법인 한국종균협회에 기탁하였다(KFCC-10750).In the above example, Corynebacterium glutamicum CS-13, in which the fermentation broth L-lysine hydrochloride concentration is improved and L-leucine is not accumulated, was finally selected and deposited in the Korean spawn association. (KFCC-10750).
선별된 코리네박테리움 CS-13, CS-14 및 모균주인 KFCC 10014를 30L 발효조에서 유가식으로 배양한후, 아미노산 조성을 분석하여 그 결과를 표6에 기재하였다.Selected Corynebacterium CS-13, CS-14 and the parent strain KFCC 10014 were cultured in a fed-batch in a 30L fermenter, and the amino acid composition was analyzed and the results are shown in Table 6.
[표 6]TABLE 6
표6. 코리네박테리움 글루타미쿰 CS-13, CS-14 및 KFCC 10014에 대한 발효조 배양결과Table 6. Fermenter Culture Results for Corynebacterium glutamicum CS-13, CS-14 and KFCC 10014
표 6으로부터 알 수 있듯이 코리네박테리움 글루타미쿰 CS-13는 배양중에 L-루이신을 전혀 첨가하지 않아도 정상배양이 가능하였다. 한편 최종배양액내 L-라이신 염산염 농도는 112g/l으로 대당수율 42% 이상이며, 발효액중의 1-루이신 축적량은 0.1g/l 이하임을 확인하였다.As can be seen from Table 6, Corynebacterium glutamicum CS-13 was able to be cultured normally without adding any L-leucine in the culture. Meanwhile, the concentration of L-lysine hydrochloride in the final culture solution was 112 g / l, the yield was 42% or more, and the accumulation amount of 1-leucine in the fermentation broth was 0.1 g / l or less.
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2000017379A2 (en) * | 1998-09-22 | 2000-03-30 | Cheil Jedang Corporation | An l-lysine-producing microorganism and a method for producing l-lysine using said microorganism |
WO2015064917A1 (en) | 2013-10-28 | 2015-05-07 | 씨제이제일제당 (주) | Microorganism of corynebacterium sp. having enhanced l-lysine producibility and method for producing l-lysine using same |
WO2015088204A1 (en) | 2013-12-13 | 2015-06-18 | 씨제이제일제당 (주) | Corynebacterium genus microorganism with improved ability to produce l-lysine and method for producing l-lysine using same |
US10179903B2 (en) | 2014-04-09 | 2019-01-15 | Cj Cheiljedang Corporation | Microorganism having L-lysine producing ability and L-lysine producing method using same |
US10526586B2 (en) | 2015-03-18 | 2020-01-07 | Cj Cheiljedang Corporation | Pyruvate dehydrogenase variants, a microorganism comprising the same and a method for producing L-amino acid using the same |
US11104925B2 (en) | 2015-07-03 | 2021-08-31 | Cj Cheiljedang Corporation | Microorganism producing L-lysine and method for producing L-lysine using the same |
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KR102277031B1 (en) * | 2021-01-29 | 2021-07-13 | 씨제이제일제당 (주) | Novel type II citrate synthase variant and a method for producing L-lysine using the same |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2000017379A2 (en) * | 1998-09-22 | 2000-03-30 | Cheil Jedang Corporation | An l-lysine-producing microorganism and a method for producing l-lysine using said microorganism |
WO2000017379A3 (en) * | 1998-09-22 | 2000-09-21 | Cheil Jedang Corp | An l-lysine-producing microorganism and a method for producing l-lysine using said microorganism |
WO2015064917A1 (en) | 2013-10-28 | 2015-05-07 | 씨제이제일제당 (주) | Microorganism of corynebacterium sp. having enhanced l-lysine producibility and method for producing l-lysine using same |
WO2015088204A1 (en) | 2013-12-13 | 2015-06-18 | 씨제이제일제당 (주) | Corynebacterium genus microorganism with improved ability to produce l-lysine and method for producing l-lysine using same |
US10179903B2 (en) | 2014-04-09 | 2019-01-15 | Cj Cheiljedang Corporation | Microorganism having L-lysine producing ability and L-lysine producing method using same |
US10526586B2 (en) | 2015-03-18 | 2020-01-07 | Cj Cheiljedang Corporation | Pyruvate dehydrogenase variants, a microorganism comprising the same and a method for producing L-amino acid using the same |
US11104925B2 (en) | 2015-07-03 | 2021-08-31 | Cj Cheiljedang Corporation | Microorganism producing L-lysine and method for producing L-lysine using the same |
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