Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of High-yielding Mutants of Streptomyces avermitilis for Avermectin B_{1a} Production through Protoplast Fusion.

원형질체 융합에 의한 Avermectina B_{1a} 고생산성 Streptomyces avermitilis 균주 개발

  • Published : 2004.06.01
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Abstract

In order to enhance the productivity of AVM $B_{la}$ 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 offusants 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 $B_{la}$ , reaching almost three times higher level (almost equal to the industrial productivity), compared to the average AVM Bla amount of the parallel mother strains.

Streptomyces avermitilis가 생산하는 이차대사산물인 avermectin $B_{la}$ (AVM $B_{la}$ /)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-isoleucine유사체인 O-methylthreoiune또는 azaleucine에 대한 저항성 변이주의 원형질체를 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 $B_{la}$ 생산성이 융합 모균주들에 비해 높게 나타났으며, 주목할 만하게도 최고 3배 정도 향상된, 거의 산업용 균주의 생산성을 갖는 균주들을 선별할 수 있었다.

Keywords

References

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