Cell Fusion Between Xylose Fermenting Yeast and Xylanase Secreting Yeast

Xylanase 분비효모와 Xylose 발효효모의 Protoplast Fusion

To improve a new yeast strain capable of converting xylan to ethanol directly, we tried protoplast fusion between xylose fermenting yeast (Candida sp. X-6-41) and xylan assimilating yeast (Crypto-coccus sp. XB-33), finally selected the most promising two fusants (XFU-1 and XFU-2). As the optimum conditions for protoplast formation, the yeast cells were cultured to exponential phase in YPD and YPX containing 0.6M KCI, respectively, and then treated with zymolyase (0.25mg/$m\ell$), cellulase(4mg/$m\ell$) and 100mM 2-mercaptoethanol at pH 8 and 3$0^{\circ}C$. The protoplasts of parental auxotrophs were fused in the presence of 20mM CaCl$_2$and 40% polyethylene glycol(M.W.4000). The physiological and morphological characteristics of the fusants, such as assimilation of carbon sources, cell size, growth rate, xylanase activity and xylan fermentation ability were investigated. Xylanase activity of fusants that cultured in chemically minimal medium was higher than that of fusants that cultured in completed medium, because xylanase producing activity of xylose fermenting yeast(X-6-41) was inhibited by isoleucine.

Xylan성 biomass로부터 직접 alcohol을 얻고자 xylose 발효효모 X-6-41 균주의 NTG mutant인 X-6-41-1(his-) 균주와 xylanase 분비효모인 XB-33 의 NTG mutant XB-33-37(Arg-) 균주를 세포융합 시켰다. 원형질체 생성조건은 KYPX(XB-33), KYPD(X-6-41)에서 대수증식기 말기까지 증식한 세포를 집균하여 zymolyase(0.25mg/$m\ell$), cellulase (4mg/$m\ell$)와 100mM 2-mercaptoethanol 처리시 protoplast화 율은 X-6-41 경우 80%, XB-33인 45%로 나타났다. 선별된 융합체의 탄소자화능은 parent의 자화능을 서로 보완하였음을 확인하였고 xylan에서의 alcohol 발효는 2% xylan에서 발효 15 일만에 총당에 대해 0.28% alcohol 농도를 나타내었다. Parent와 fusant의 xylanase 활성은 완전배지 보다 최소배지상에서 높았는데 이것은 xylose 발효효모(X-6-41)의 경우 isoleucine에 의한 xylanase production inhibition 효과와 일치하였다.