원형질체 융합에 의한 Avermectin B1a 고생산성 Streptomyces avermitilis 균주 개발
Development of High-yielding Mutants of Streptomyces avermitilis for Avermectin B1a Productionthrough Protoplast Fusion.
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Streptomyces avermitilis가 생산하는 이차대사산물인 avermectin B1a(AVM B1a)의 생산성을 향상시키고자, 고생산성 균주를 융합파트너로 이용하여 원형질체 융합에 필요한 기본 실험조건을 확립하였고, 대량 선별시스템을 이용하여 다양한 융합균주들을 선별하였다. 특별한 표지인자가 없는 경우에도 원형질체 융합에 의해 유전자재조합체들을 선별할 수 있는 방법을 개발하였다. 즉 고생산성 균주의 원형질체(L-isoleucine 유사체인 O-methylthreonine 또는 azaleucine에 대한 저항성 변이주의 원형질체)의 치사율이 약 95% 정도 되도록 UV나 NTG로 각 원형질체를 돌연변이시킨 후, 융합을 시도하여 재생된 변이주를 원형질 융합된 유전자재조합체로 간주하는 방법을 고안하였다. 돌연변이원으로 UV를 이용할 경우 대부분의 유전자재조합 균주들의 AVM B1a 생산성이 융합 모균주들에 비해 높게 나타났으며, 주목할 만하게도 최고 3배 정도 향상된, 거의 산업용 균주의 생산성을 갖는 균주들을 선별할 수 있었다. 감사의 글
Abstract
In order to enhance the productivity of AVM B1a produced by Streptomyces avermitilis as a secondary metabolite, we established a basic protocol necessary for protoplast fusion with high-producing strains as a fusion partner, and then obtained various kinds of fusants by adopting a massive strain-development procedure (a miniaturized strain screening system). An alternative fusion method using UV and/or NTG mutation of protoplasts was developed to screen genetic recombinants without specific selectable markers. In this method, the mutants obtained by protoplast fusion after UV and/or NTG treatment (95% death rate) of the respective fusion partner (protoplasts of the respective mutants resistant against L-isoleucine antimetabolites such as O-methylthreonine and/or azaleucine) were regarded as DNA-recombined protoplast fusants. Notably it was demonstrated that most of the protoplast recombinants obtained by the UV mutation method were able to biosynthesize higher amount of AVM B1a, reaching almost three times higher level (almost equal to the industrial productivity), compared to the average AVM B1a amount of the parallel mother strains.
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