KR100655902B1 - Corynebacterium ammoniagenes cjxcv24 kccm-10693p having high activity of convertion of xmp to gmp - Google Patents
Corynebacterium ammoniagenes cjxcv24 kccm-10693p having high activity of convertion of xmp to gmp Download PDFInfo
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Abstract
Description
본 발명은 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530의 변이주로서, XMP에서 GMP로 전환율이 향상된 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P에 관한 것이다.The present invention relates to a variant strain of Corynebacterium ammonia genes CJXFT0301 KCCM-10530, and relates to Corynebacterium ammonia genes CJXCV24 KCCM-10693P with improved conversion from XMP to GMP.
GMP(5’-guanine monophosphate)는 IMP(inosine 5’-monophosphate)와 더불어 식품 조미 첨가제로 널리 이용되고 있는 물질이다. GMP는 그 자체로 버섯 맛을 내는 것으로 알려져 있으나, 이는 주로 모노소디움 글루탐산(MSG)의 풍미를 강화하는 것으로 알려져 있다. 이러한 성질은 특히 5’-이노신산과 같이 쓰여졌을 때 강하게 나타난다.GMP (5'-guanine monophosphate) is a widely used food seasoning additive along with IMP (inosine 5'-monophosphate). GMP is known to flavor mushrooms by itself, but it is known to enhance the flavor of monosodium glutamic acid (MSG) primarily. This property is particularly strong when used with 5'-inosinic acid.
지금까지 알려진 GMP의 제조방법은 첫째, 효모세포로부터 추출한 리보핵산(RNA)를 효소학적으로 분해하는 방법, 둘째, 미생물 발효법으로 5’-구아닐산을 직접 발효하는 방법, 셋째, 미생물 발효법으로 생산한 구아노신을 화학적으로 인산화 시키는 방법, 넷째, 미생물 발효법으로 생산한 구아노신을 효소적 방법으로 인산화 시키는 방법, 다섯째, 미생물 발효법으로 생산한 XMP를 코리네형 미생물을 이용하여 5’-구아닐산으로 전환하는 방법, 여섯째, 미생물 발효법으로 생산한 XMP(5′-xanthosine monophosphate)를 대장균을 이용하여 GMP로 전환시키는 방법을 들 수 있다. 상기 6가지의 방법 중에서 첫번째의 방법은 원료 수급 및 경제성에 문제가 있으며, 두번째의 방법은 GMP의 세포막 투과성의 문제로 인하여 수율이 낮다는 단점이 있어 그 외의 방법이 공업적으로 주로 이용되고 있다. 특히, XMP를 효소적으로 전환시키는 방법이 널리 이용되고 있다.Known methods for manufacturing GMP include first, enzymatically degrading ribonucleic acid (RNA) extracted from yeast cells, second, direct fermentation of 5'-guanylic acid by microbial fermentation, and third, guano produced by microbial fermentation. Chemically phosphorylation of god, fourth, enzymatically phosphorylated guanosine produced by microbial fermentation method, fifth, conversion of XMP produced by microbial fermentation method to 5'-guanylic acid using coryneform microorganism, sixth, XMP (5′-xanthosine monophosphate) produced by the microbial fermentation method using E. coli to convert to GMP. Among the six methods, the first method has a problem in supply and demand of raw materials, and the second method has a disadvantage in that the yield is low due to the problem of GMP cell membrane permeability, and other methods are mainly used industrially. In particular, a method for enzymatically converting XMP is widely used.
종래 XMP의 제조방법에는 화학합성법, 또는 효모 중의 리보핵산을 분해하여 제조된 GMP를 탈 아미노화 하는 제조법, 그리고 발효법으로는 발효배지 내 전구물질로 크산틴(Xanthine)을 첨가하는 방법과 미생물 변이주에 의한 제조법, 항생물질 첨가에 의한 제조법(일본특허 소42-1477, 소 44-20390) 및 계면활성제 첨가에 의한 제조법(일본특허 소42-3825, 소42-3838) 등이 알려져 있다. Conventional XMP production methods include chemical synthesis or deamination of GMP prepared by decomposing ribonucleic acid in yeast, and fermentation methods add xanthine as a precursor in fermentation broth and microbial mutants. The manufacturing method by the addition of antibiotics, the manufacturing method by adding antibiotics (Japanese Patent No. 42-1477, the 4444390), the manufacturing method by adding surfactant (Japanese Patent No. 42-3825, Small 42-3838), etc. are known.
상기의 방법 중에서도 미생물 변이주에 의한 XMP의 직접적인 발효 제조 방법이 공업적으로 유리하다. XMP를 직접 발효하고 대장균의 효소(5’-크산틸산 아미나아제)를 이용하여 GMP로 전환하는 반응식은 하기와 같다. Among the above methods, a method of directly producing fermentation of XMP by microbial mutants is industrially advantageous. The reaction scheme of directly fermenting XMP and converting it to GMP using an E. coli enzyme (5′-xanthyl acid aminase) is as follows.
XMP + ATP + NH4 + → GMP + AMP + PPiXMP + ATP + NH 4 + → GMP + AMP + PPi
상기의 반응식에 있어서, XMP 및 ATP는 XMP 생산 변이체에서 제공되고 NH4 +는 외부에서 첨가하며 XMP 아미나아제는 대장균에서 과발현하여 사용한다. In the above scheme, XMP and ATP are provided in XMP producing variants, NH 4 + is added externally and XMP aminase is overexpressed in E. coli.
GMP의 생산성을 높이기 위해서는 XMP를 과생산할 뿐 아니라 XMP에서 GMP으로 의 전환반응이 효과적으로 이루어져야 한다. 이것은 생성된 GMP의 분해 또는 다른 물질로의 전환이 적어야 하며, 전환반응시 필요한 ATP의 재생이 원활히 이루어지기 충분한 양의 균체가 있어야 한다는 것을 의미한다. In order to increase the productivity of GMP, not only XMP is over-produced, but also the conversion reaction from XMP to GMP should be effective. This means that there should be little degradation of the resulting GMP or conversion to other substances, and there should be enough cells to facilitate the regeneration of the ATP required for the conversion reaction.
코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530는 과량의 XMP를 생산할 수 있었으나 GMP로의 낮은 전환율을 보였다. Corynebacterium ammonia genes CJXFT0301 KCCM-10530 was able to produce excess XMP but showed a low conversion to GMP.
XMP에서 GMP로의 전환율을 향상시키기 위해 기존 균주의 형질을 개량하여 GMP의 분해 및 전환 활성이 약해지고 균체성장이 향상된 특징을 지닌 새로운 균주의 개발이 필요하다. In order to improve the conversion rate from XMP to GMP, it is necessary to develop a new strain having characteristics of weakening degradation and conversion activity of GMP and improving cell growth by improving the traits of existing strains.
이에 따라, 본 발명자들은 기존 균주인 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530에 변이를 유발하여 변이주를 선별하고, 이의 XMP에서 GMP로의 높은 전환율 및 GMP 자체의 분해 또는 전환율이 낮은 균주를 확인함으로서 본 발명을 완성하였다.Accordingly, the present inventors select the mutant strains by causing a mutation in the existing strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530, by identifying a strain having a high conversion rate from XMP to GMP and low degradation or conversion of GMP itself The present invention has been completed.
본 발명의 목적은 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530의 변이주로서, XMP에서 GMP로 전환율이 향상된 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P을 제공하며, 또한 고수율로 GMP를 생산하는 방법을 제공하고자 한다.It is an object of the present invention to provide a modified strain of Corynebacterium ammonia genes CJXFT0301 KCCM-10530, which provides improved Corynebacterium ammonia genes CJXCV24 KCCM-10693P, which produces GMP with high yield. To provide a method.
상기 목적을 달성하기 위하여, 본 발명은 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530의 변이주로서, XMP(5’-xanthosine monophosphate)에서 GMP (5’-guanine monophosphate)로 전환율이 향상된 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P을 제공한다.In order to achieve the above object, the present invention is a variant of Corynebacterium ammonia genes CJXFT0301 KCCM-10530, Corynebacterium with improved conversion rate from 5'-xanthosine monophosphate (GMP) to 5'-guanine monophosphate (GMP) Ammonia Genes CJXCV24 KCCM-10693P is provided.
또한, 상기 변이주를 이용하여 GMP를 생산하는 방법을 제공한다.In addition, it provides a method for producing GMP using the mutant strain.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530는 과량의 XMP를 생산할 수는 있으나, GMP로의 낮은 전환율로 인하여 GMP 생산에는 부적합한 균주이다. Corynebacterium ammonia genes CJXFT0301 KCCM-10530 is capable of producing excess XMP, but is unsuitable for GMP production due to its low conversion to GMP.
따라서, 상기 균주를 친주로 하여 N-메틸 -N`-니트로 -N-니트로소구아니딘(NTG) 등의 변이유발제를 사용하여 통상적인 방법에 따라 변이를 유발한 후, 변이체 라이브러리를 제작하고, 최소배지에 프롤린유사체 및 글루타민유사체를 첨가한 배지에 도말하여 생성된 콜리니들을 확보한다.Therefore, using the above strain as a parent strain, using a mutagenesis agent such as N-methyl-N`-nitro-N-nitrosoguanidine (NTG) to induce mutations according to a conventional method, and then prepare a variant library, The colonies produced by smearing on a medium to which proline and glutamine analogs are added to the medium are secured.
상기 콜로니들은 고속선별법(High-throughput screening)을 이용하여 XMP에서 GMP로의 전환율을 우수한 콜로니만을 선별할 수 있다.The colonies can select only colonies having excellent conversion rate from XMP to GMP using high-throughput screening.
상기 고속선별법을 확립하기 위해 마이크로플레이트에서의 성장 조건을 설계할 수 있는데, 비교 실험을 위해 먼저 야생주와 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530를 비교균으로 사용하여 같은 균에 대한 성장이 가장 균일하게 되는 조건을 접종 방법, 배양 시간 등을 조절하여 설정하고, 그 결과 오차 범위가 10% 미만의 배양 배지 및 방법 조건을 찾을 수 있다.To establish the high-speed screening method, growth conditions in a microplate may be designed. For comparison experiments, wild strains and Corynebacterium ammonia genes, CJXFT0301 KCCM-10530, were used as comparative bacteria. The most uniform conditions are set by adjusting the inoculation method, incubation time, and the like, and as a result, the culture medium and method conditions having an error range of less than 10% can be found.
또한, GMP분해도 측정을 위해 배양이 끝난 후 첨가물과 온도, 반응시간 등을 조절하여 야생주와 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530의 차이가 가장 많이 나는 조건을 확립할 수 있다. In addition, by adjusting the additives, temperature, reaction time, etc. after the end of the cultivation to determine the GMP degradation degree can be established conditions that the difference between the wild wine and Corynebacterium ammonia genes CJXFT0301 KCCM-10530.
상기 고속선별법을 사용하여 선별된 변이주를 코리네박테리움 암모니아게네스 CJXCV24라 명명하고, 이를 한국미생물보존센터에 기탁하였고, 기탁번호는 KCCM-10693P이다.The mutant strains selected using the high-speed screening method were named Corynebacterium ammonia genes CJXCV24, which was deposited with the Korea Microorganism Conservation Center, and the accession number is KCCM-10693P.
본 발명의 변이주인 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P는 직접발효법에 의해 XMP를 고농도, 고수율로 생산하면서도, 세포성장이 향상되어 XMP에서 GMP로의 전환율이 탁월하게 향상되고, 전환반응시간이 현저히 단축되어 GMP를 고수율로 생산할 수 있는 변이주이다. 또한, 생산된 GMP를 분해 또는 전환시키는 활성은 저해되어 생산된 GMP를 효과적으로 수득할 수 있는 이점이 있다.Corynebacterium ammonia genes CJXCV24 KCCM-10693P, a mutant of the present invention, produced high concentrations and high yields of XMP by direct fermentation, and improved cell growth, resulting in an excellent conversion of XMP to GMP, and a conversion reaction time. This is a markedly shortened mutant that can produce high yields of GMP. In addition, the activity of degrading or converting the produced GMP is inhibited, there is an advantage that can effectively obtain the produced GMP.
또한, 본 발명의 변이주인 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P는 모균주인 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530 균주의 특징인 아데닌 요구성, 구아닌 요구성, 라이소자임 감수성, 프롤린유사체 내성, 글루타민유사체 내성 및 트립토판유사체 내성을 동일하게 보유한다.In addition, the variant strain Corynebacterium ammonia genes CJXCV24 KCCM-10693P of the present invention is characterized by adenine requirements, guanine requirements, lysozyme susceptibility, proline analogs of the parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530 strain Resistance, glutamine-like resistance, and tryptophan-like resistance are the same.
따라서, 본 발명은 상기 변이주를 이용하여 GMP를 생산하는 방법을 제공한다.Therefore, the present invention provides a method for producing GMP using the mutant strain.
이하, 본 발명을 하기의 실시예 및 실험예에 의하여 더욱 상세히 설명한다. 다만, 하기의 실시예 및 실험예는 본 발명의 예시일 뿐, 본 발명이 이에 의하여 한정되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only examples of the present invention, and the present invention is not limited thereto.
참조예Reference Example 1. 배지 조성 및 조건 1. Medium composition and condition
1-1. 1-1. 최소배지Minimum badge
최소배지는 포도당 20g/L, 인산제1칼륨 1g/L, 인산제2칼륨 1g/L, 우레아 2g/L, 황산암모늄 3g/L, 황산마그네슘 1g/L, 염화칼슘 100mg/L, 황산철 20mg/L, 황산망간 10mg/L, 황산아연 10mg/L, 비오틴 30ug/L, 티아민산염 0.1mg/L, 황산구리 0.8mg/L, 아데닌 20mg/L, 구아닌 20mg/L으로 구성되며 pH 7.2로 조정하였다.The minimum medium is glucose 20g / L, 1g / L potassium phosphate, 1g / L dipotassium phosphate, 2g / L urea, 3g / L ammonium sulfate, 1g / L magnesium sulfate, 100mg / L calcium chloride, 20mg / iron sulfate L, manganese sulfate 10mg / L, zinc sulfate 10mg / L, biotin 30ug / L, thiamine salt 0.1mg / L, copper sulfate 0.8mg / L, adenine 20mg / L, guanine 20mg / L and adjusted to pH 7.2.
1-2. 1-2. 프롤린유사체Proline analogue 및 And 글루타민유사체Glutamine analogue 첨가배지Medium added
상기 참조예 1-1의 최소배지에 프롤린유사체 100mg/L, 글루타민유사체 100mg/L를 각각 첨가하여 배지를 조성하였다.A medium was prepared by adding 100 mg / L of proline analog and 100 mg / L of glutamine analog to the minimum medium of Reference Example 1-1, respectively.
실시예Example 1. 변이주 1. Variation 코리네박테리움Corynebacterium 암모니아게네스Ammonia Genes CJXCV24CJXCV24 KCCMKCCM 제작 making
1-1. 1-1. 변이체Variant 라이브러리 제작 Library Authoring
5’-구아닐산(GMP) 저분해 또는 저전환 변이체를 수득하기 위하여 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530를 모균주로 사용하고, 변이 유발제인 N-메틸 -N`-니트로 -N-니트로소구아니딘(NTG)을 사용한 통상적인 방법에 따라 변이체 라이브러리를 제작하였다. 그 후, 코리네박테리움 암모니아게네스 CJXFT0301 KCCM 10530을 선별한 배지인 상기 참조예 1-2의 프롤린유사체 및 글루타민유사체 첨가배지에 도말 하여 생성된 콜로니들을 확보하였다. Corynebacterium ammonia genes CJXFT0301 KCCM-10530 was used as a parent strain to obtain 5′-guanylic acid (GMP) low resolution or low conversion variants, and the mutation inducer N-methyl-N′-nitro-N-nitro Variant libraries were prepared according to conventional methods using soguanidine (NTG). Thereafter, the colonies produced by smearing the corynebacterium ammonia genes CJXFT0301 KCCM 10530 on the medium containing the proline and glutamine analogs of Reference Example 1-2 were selected.
1-2. 고속선별법(High-throughput screening) 조건 확립1-2. Establish high-throughput screening conditions
상기에서 생성된 콜로니들 중에서 XMP에서 GMP로의 전환율이 높은 변이주를 선별할 목적으로 고속선별법 조건을 확립하기 위해 마이크로플레이트(micro-plate)에서의 성장 조건을 설계하였다. Growth conditions in a micro-plate were designed to establish high-speed screening conditions for the purpose of selecting mutants with high conversion rates from XMP to GMP among the colonies generated above.
비교 실험을 위해 먼저 야생주와 코리네박테리윰 암모니아게네스 CJXFT0301 KCCM-10530를 비교균으로 사용하여 같은 균에 대한 성장이 가장 균일하게 되는 조건으로 접종 방법, 배양 시간 등을 조절하여 설정한 후, 오차 범위가 10% 미만의 배양 배지 및 방법 조건을 확립하였다.For comparative experiments, wild seed and Corynebacterium ammonia genes CJXFT0301 KCCM-10530 were used as comparative bacteria. Culture media and method conditions with an error range of less than 10% were established.
그 결과, 마이크로플레이트용 배지는 포도당 10g/L, 펩톤 10g/L, 효모엑기스 5g/L, 염화나트륨 2.5g/L, 우레아 3g/L, 아데닌 200mg/L 및 구아닌 200mg/L로 구성되며, pH 7.2로 조정하는 것이 가장 우수하였으며, 배양 조건은 Deep wellTM(Bioneer)에 상기 배지 1㎖씩 분주하고, 균을 접종하여 MegaGrowTM(Bioneer)에 2일 동안 30℃, 500rpm에서의 배양조건을 확립하였다.As a result, the medium for microplates consisted of 10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 2.5 g / L sodium chloride, 3 g / L urea, 200 mg / L adenine and 200 mg / L guanine, pH 7.2 The culture conditions were best adjusted, and the culture conditions were divided into 1 ml of the medium in Deep well TM (Bioneer), and inoculated with the bacteria to incubate the culture conditions at 30 ° C. and 500 rpm for 2 days in MegaGrow TM (Bioneer). .
또한, GMP분해도 측정을 위해 배양이 끝난 후 첨가물과 온도, 반응시간 등을 조절하여 야생주와 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530의 차이가 가장 많이 나는 조건을 확립하였는바, 그 결과는 첨가물 조성은 피틴산(phytic acid) 1.8g/L, MgSO4 4.8g/L, 니민(Nymeen) 3mL/L, 아데닌(adenine) 100mg/L, Na2HPO4 7.7g/L, 글루코스 46g/L으로, 반응 조건의 경우에는 상기 첨가물 200ul 첨 가하여 42℃, 500rpm의 조건하에서 4시간 반응시켜, 반응물 측정 조건은 배양액을 원심 분리하여 상등액만을 취해 50배 희석한 후 UV파장인 Abs. 290nm에서 반응 전과 4시간 반응 후의 값을 측정하여 비교하는 것으로 하였다.In addition, after the incubation, the additives, temperature, and reaction time were adjusted to determine the GMP degradation level. Additive composition was 1.8 g / L phytic acid, 4.8 g / L MgSO 4 , 3 mL / L Nimine, 100 mg / L adenine, 7.7 g / L Na 2 HPO 4 , 46 g / L glucose In the case of reaction conditions, the additive was added 200ul and reacted for 4 hours under the conditions of 42 ° C and 500rpm, and the reaction conditions were measured by centrifuging the culture solution, taking only the supernatant, diluting 50 times, and then absorbing the UV wavelength of Abs. The value before and after 4 hours of reaction at 290 nm was measured and compared.
1-3. 변이주 1-3. Mutant 코리네박테리움Corynebacterium 암모니아게네스Ammonia Genes CJXCV24CJXCV24 KCCMKCCM 제작 making
상기 실시예 1-1에서 제작한 변이체 콜로니 라이브러리를 상기 실시예 1-2에서 확립한 고속선별법의 조건으로서, 마이크로플레이트용 배지(포도당 10g/L, 펩톤 10g/L, 효모엑기스 5g/L, 염화나트륨 2.5g/L, 우레아 3g/L, 아데닌 200mg/L 및 구아닌 200mg/L, pH 7.2)에 도말하여, Deep wellTM(Bioneer)에 상기 배지 1mL씩 분주하고, 균을 접종하여 MegaGrowTM(Bioneer)에 2일 동안 30℃, 500rpm에서의 배양하였다.As a condition of the high-speed screening method in which the variant colony library prepared in Example 1-1 was established in Example 1-2, microplate medium (glucose 10g / L, peptone 10g / L, yeast extract 5g / L, sodium chloride) 2.5 g / L, urea 3 g / L, adenine 200 mg / L and guanine 200 mg / L, pH 7.2), dispense 1 mL of the medium into Deep well TM (Bioneer), inoculate the bacteria and inoculate the MegaGrow TM (Bioneer) Incubated at 30 ° C., 500 rpm for 2 days.
그 후, 상기 실시예 1-2에서 확립한 첨가물(피틴산 1.8g/L, MgSO4 4.8g/L, 니민(Nymeen) 3mL/L, 아데닌(adenine) 100mg/L, Na2HPO4 7.7g/L, 글루코스 46g/L)을 200ul 첨가하여 42℃, 500rpm의 조건하에서 4시간 동안 전환반응시킨 후, 상기 배양액을 원심 분리하여 상등액만을 취해 50배 희석한 후 UV파장인 Abs. 290nm에서 반응 전과 4시간 반응 후의 값을 측정하여 비교함으로서 5’-구아닐산(GMP)의 반응 후 농도가 가장 높은 콜로니를 선별하였다. Subsequently, the additives established in Examples 1-2 (1.8 g / L phytic acid, 4.8 g / L MgSO 4 , 3 mL / L Nimine, 100 mg / L adenine, and 7.7 g / Na 2 HPO 4). L, glucose 46g / L) was added 200ul and the conversion reaction for 4 hours under the conditions of 42 ℃, 500rpm, the culture solution was centrifuged to take only the supernatant and diluted 50-fold Abs. The colonies having the highest concentration after the reaction of 5'-guanylic acid (GMP) were selected by measuring and comparing the values before and after the reaction for 4 hours at 290 nm.
그 후, 상기에서 선별된 콜로니들을 하기와 같은 방법으로 삼각플라스크 발 효를 수행하였다.Thereafter, the colonies selected above were fermented with a Erlenmeyer flask in the following manner.
종배지(포도당 30g/L, 펩톤 15g/L, 효모엑기스 15g/L, 염화나트륨 2.5g/L, 우레아 3g/L, 아데닌 150mg/L, 구아닌 150mg/L, pH 7.2) 5㎖을 지름 18mm 시험관에 분주하고 상법에 따라 가압 살균한 후 사용 균주를 접종하고 180rpm으로 30℃에서 18시간 진탕 배양하여 종배양액으로 사용하였다. 5 ml of seed medium (30 g / L glucose, 15 g / L peptone, 15 g / L yeast extract, 2.5 g / L sodium chloride, 3 g / L urea, 150 mg / L adenine, guanine 150 mg / L, pH 7.2) was placed in an 18 mm diameter test tube. After dispensing and autoclaving according to the conventional method, the strain was inoculated and the culture was shaken for 18 hours at 30 ° C. at 180 rpm to be used as a seed culture solution.
발효배지 중 본배지(포도당 60g/L, 황산마그네슘 10g/L, 황산철 20mg/L, 황산아연 10mg/L, 황산망간 10mg/L, 아데닌 30mg/L, 구아닌 30mg/L, 비오틴 100ug/L, 황산구리 1mg/L, 티아민염산염 5mg/L, 염화칼슘 10mg/L, pH 7.2)와 별살배지(인산제1칼륨 10g/L, 인산제2칼륨 10g/L, 우레아 7g/L, 황산암모늄 5g/L)를 각각 상법에 따라 가압 살균하여 미리 가압 살균한 500mL 용량의 진탕용 삼각 플라스크에 29mL과 10mL 씩 분주하고 종배양액 1mL을 식균한 다음 30℃에서, 200rpm의 회전수로 90시간 동안 배양하였다.Main medium in fermentation medium (glucose 60g / L, magnesium sulfate 10g / L, iron sulfate 20mg / L, zinc sulfate 10mg / L, manganese sulfate 10mg / L, adenine 30mg / L, guanine 30mg / L, biotin 100ug / L, Copper sulfate 1mg / L, thiamine hydrochloride 5mg / L, calcium chloride 10mg / L, pH 7.2) and starch medium (10 g / L potassium phosphate, 10 g / L potassium phosphate, urea 7 g / L, ammonium sulfate 5 g / L) After autoclaving according to the conventional method, 29mL and 10mL were respectively dispensed into a 500mL shake-type Erlenmeyer flask previously autoclaved, and 1mL of the culture medium was inoculated, and then incubated at 30 ° C. with a rotation speed of 200 rpm for 90 hours.
상기 배양 완료한 후, 모균주와 비교하여 5’-크산틸산의 농도가 비슷하거나 높은 것, 세포 농도가 증가한 변이체를 최종 선별하여 코리네박테리움 암모니아게네스 CJXCV24라 명하고, 이를 2005년 11월 9일자에 한국미생물보존센터에 기탁하였고, 그 기탁번호는 KCCM-10693P이다. After completion of the incubation, the concentration of 5'-xanthyl acid in comparison with the parent strain was higher or higher, the final selection of the variants with increased cell concentration was named Corynebacterium ammonia genes CJXCV24, November 2005 On 9th, it was deposited with the Korea Center for Microbiological Conservation and its accession number is KCCM-10693P.
5’-크산틸산의 배지내 축적량은 모균주인 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530가 30.1 g/L이며, 본 발명의 변이주 코리네박테리움 암모니아게네스 CJCV24 KCCM-10693P의 경우에는 31.2g/L임을 확인할 수 있었다(5’-크산틸 산의 축적 농도는 5’-크산틸산 나트륨·7H2O로 표시하였다). 세포농도는 100배 희석하여 Abs.562nm에서 측정한 결과 기존 균주가 약 0.3, 본 발명의 변이주가 0.4로 약 30% 이상의 성장성 향상을 보였다. The accumulation amount of 5'-xanthyl acid in the medium was 30.1 g / L for the parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530, and 31.2 for the variant strain Corynebacterium ammonia genes CJCV24 KCCM-10693P of the present invention. g / L (the accumulation concentration of 5'-xanthyl acid was expressed by 5'-sodium xanthyl acid 7H 2 O). Cell concentration was diluted 100-fold and measured at Abs. 562 nm, the existing strain was about 0.3, the mutant strain of the present invention was 0.4, showing a growth improvement of about 30% or more.
실험예Experimental Example 1. 5′-구아닐산( 1.5 'guanylic acid ( GMPGMP )으로의 전환반응Conversion to
5’-구아닐산(GMP)으로의 전환 반응은 5’-크산틸산(XMP) 삼각플라스크 발효 액 40mL에 상기 실시예 1-2에서 확립한 첨가물(피틴산 1.8g/L, MgSO4 4.8g/L, 니민(Nymeen) 3mL/L, 아데닌(adenine) 100mg/L, Na2HPO4 7.7g/L, 글루코스 46g/L) 2.8mL과 XMP 아미나아제를 생산하는 대장균 발효액 4.2mL을 첨가하고 42℃, 500rpm의 조건하에서 24시간 동안 반응시킴으로 전환반응을 수행하였다.The conversion reaction to 5'-guanylic acid (GMP) was carried out in 40 mL of 5'-xanthylic acid (XMP) Erlenmeyer flask fermentation broth. (Nymeen) 3 mL / L, adenine 100 mg / L, Na2HPO4 7.7 g / L, glucose 46 g / L) and 2.8 mL of E. coli fermentation broth producing XMP aminase were added at 24 ° C. at 42 ° C. and 500 rpm. The reaction was carried out by reacting for a time.
상기 실험 수행의 결과, 모균주인 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530는 전환 반응이 24시간 소요되었으나 본 발명의 변이주인 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P는 15시간이 사용되어 9시간의 시간 단축 효과를 확인할 수 있었다. As a result of the experiment, the parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530 took a conversion reaction 24 hours, but the variant strain Corynebacterium ammonia genes CJXCV24 KCCM-10693P was used for 15 hours 9 hours of time reduction effect was confirmed.
또한 최종 생성된 5’-구아닐산(GMP)의 농도는 모균주가 22.5g/L로 75%의 전환율을 보였고, 본 발명의 변이주인 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P는 26.4g/L로 83%의 전환율을 보임을 확인할 수 있었다. In addition, the final concentration of 5'-guanylic acid (GMP) showed a conversion rate of 75% to 22.5g / L of the parent strain, Corynebacterium ammonia genes CJXCV24 KCCM-10693P is 26.4g / L The conversion rate was 83%.
상술한 바와 같이, 본 발명은 코리네박테리움 암모니아게네스 CJXFT0301 KCCM-10530의 변이주로서, XMP에서 GMP로 전환율이 향상된 코리네박테리움 암모니아게네스 CJXCV24 KCCM-10693P를 제공한다.As described above, the present invention provides a variant of Corynebacterium ammonia genes CJXFT0301 KCCM-10530, Corynebacterium ammonia genes CJXCV24 KCCM-10693P with improved conversion from XMP to GMP.
본 발명의 변이주에 의할 경우, 직접발효법에 의해 XMP를 고농도, 고수율로 생산하면서도, 세포성장이 향상되어 XMP에서 GMP로의 전환율이 탁월하게 향상되고, 전환반응시간이 현저히 단축되어 전환반응속도가 상승되어 빠른시간내에 GMP를 고수율로 생산할 수 있는 효과를 가진다. 또한, 생산된 GMP를 분해 또는 전환시키는 활성은 저해되어 생산된 GMP를 효과적으로 수득할 수 있는 이점이 있다.According to the mutant strain of the present invention, while producing high concentration and high yield of XMP by the direct fermentation method, the cell growth is improved and the conversion rate from XMP to GMP is excellently improved, and the conversion reaction time is markedly shortened so that the conversion reaction rate is increased. It is elevated and has the effect of producing GMP with high yield in a short time. In addition, the activity of degrading or converting the produced GMP is inhibited, there is an advantage that can effectively obtain the produced GMP.
또한, 본 발명에서 이용한 고속선별법(High-throughput screening)을 이용할 경우, 세포 외부의 GMP를 분해하거나 소모하는 활성은 낮으나 세포 농도가 높은 변이주만을 선택적으로 선별할 수 있다.In addition, when using the high-throughput screening method used in the present invention, the activity of degrading or consuming GMP outside the cell is low, but the cell concentration is high. Only mutant strains can be selectively screened.
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| WO2023048343A1 (en) | 2021-09-23 | 2023-03-30 | 씨제이제일제당 (주) | Novel glutamine hydrolysis gmp synthase variant and method for producing purine nucleotides using same |
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