Intraspecific Protoplast Fusion of Citric Acid Producer, Candida lipolytica

구연산 생성 Candida lipolytica의 원형질체 융합

In order to develope a protoplast fusion system for citric acid and SCP producing Candida lipolytica, the optimal conditions for the formation and regeneration of protoplast were examined and the protoplast fusion was performed. At the optimal conditions of growth phase and Zymolyase treatment, frequencies of protoplast formation were 98%. Approximately 20-30% of protoplasts were regenerated on the regeneration minimal medium containing 3% agar and 30mM $CaCl_2$ with the overlay of the same medium. The fusion frequencies, 4-5${\pm}$10$^{-4}$, were accomplished by the treatment of two nutritionally complementary auxotrophic protoplasts, L-14 ($lys^-$) and T-24 (X$30^-$), with 30% PEG 6000 containing 100mM $CaCl_2$ at $30^{\circ}C$ for 20 minutes.

내산성이 우수하고 citric acid와 SCP를 많이 생산할 수 있는 효모균주를 육종하기 위하여 구연산 생성효모인 Candide lipolytica의 종내 원형 질체융합 조건을 검토하였다. 배양기간과 효소처리조건을 최적화함에 의해 98%의 protoplast가 형성되었다. 3 % agar와 30mM $CaCl_2$를 함유한 재생용최소배지에 동일배지의 중층에 의해 약 20-30%의 protoplast가 재생되었다. 2개의 영양요구성이 상보적인 영양요구성원형질체, L-14($lys^-$)와 T-24($try^-$)에 100mM $CaCl_2$를 함유하는 30% PEG 6000을 $30^{\circ}C$ 에서 20분간 처리하여 4-5${\pm}$$10^{-4}$의 융합빈도가 얻어졌다.